bluuebunny/crossref_metadata_2025 huggingface dataset:
Potassium depletion and hypertonic medium reduce “non‐coated” and clathrin‐coated pit formation, as well as endocytosis through these two gates
Authors: Carpentier Jean‐Louis , Sawano Fumio , Geiger Dominique , Gorden Phillip , Perrelet Alain , Orci Lelio
Published: February 2005

Intracellular potassium depletion inhibits receptor‐mediated endocytotic processes occurring through clathrin‐coated pits. Besides the clathrin‐coated pit route, flask‐shaped invaginations that do not bear a typical clathrin coat have been recently implicated in receptor‐mediated endocytosis of cholera toxin. These invaginations are called “non‐coated” to distinguish them from the typical clathrin‐coated pits. In the present study, we have investigated whether “non‐coated” invaginations are sensitive, as are clathrin‐coated pits, to potassium depletion and whether hypertonic medium, which inhibits receptor‐mediated endocytosis, also affects “non‐coated” invaginations. We found that 1) both potassium depletion and hypertonic medium reduce “non‐coated” invaginations on the cell surface; 2) similar to potassium depletion, hypertonic medium markedly decreases the number of clathrin‐coated pits; 3) these changes are accompanied by an inhibition of the internalization (measured morphologically) of cholera toxin‐gold through “non‐coated” invaginations, as well as of α2‐macroglobulin‐gold taken up by clathrin‐coated pits; and 4) in addition, both the hypertonic medium and potassium depletion inhibit the uptake of horseradish peroxidase, a marker of fluid‐phase endocytosis.
Ionic dependence of glycylsarcosine uptake by isolated chicken enterocytes
Authors: Calonge M. L. , Ilundain A. , Bolufer J.
Published: February 2005

Dipeptide transport was studied in chicken enterocytes and its properties compared with those of Na+‐dependent sugar transport. Results showed that 1) isolated cells were capable of accumulating glycylsarcosine (Gly‐Sar) against a concentration gradient (2.5‐ to 3.0‐fold accumulation). This uptake was maximal at pH 6.0, and it was inhibited by Na+‐free medium and by ouabaine; 2) uptake of Gly‐Sar was not affected by methionine and was competitively inhibited by carnosine, with a Ki of 12 mM; 3) the protonophore FCCP inhibited both Gly‐Sar and 3‐oxy‐methyl‐D‐glucose (3‐OMG) uptake by the cells; 4) amiloride, a well‐known inhibitor of the Na+/H+ exchanger system stimulated 3‐OMG uptake and inhibited Gly‐Sar uptake, its effects being greater at pH 7.4; 5) and monensine prevents the effects of amiloride on both sugar and dipeptide uptake. In summary, Gly‐Sar uptake depends on extracellular Na+ in an indirect manner via its effect on H+ efflux, and it appears to be dependent on an inward H+ gradient.
Effects of 1,25‐dihydroxyvitamin D3 on cell‐cycle kinetics of T 47D human breast cancer cells
Authors: Eisman John A. , Sutherland Robert L. , McMenemy M. Lynne , Fragonas Jean‐Charles , Musgrove Elizabeth A. , Pang Grace Y. N.
Published: February 2005

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25‐dihydroxyvitamin D3 [1,25‐(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25‐(OH)2D3, which inhibit cellular replication, on the cell‐cycle kinetics of a 1,25‐(OH)2D3‐responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25‐(OH)2D3 in the range 10−9 M to 10−6 M caused a time‐ and concentration‐dependent decrease in cell numbers. Treatment of cells growing in charcoal‐treated fetal calf serum with 10−8 M 1,25‐(OH)2D3 for 6 days reduced cell numbers to 49% ± 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% ± 0.5% (n = 11) to 19.6% ± 2.3% (n = 9), significant by paired analysis (P > 0.002). At higher concentrations of 1,25‐(OH)2D3 (10−7 −10−6 M), there was a concentration‐dependent decline in S phase and increases in both Go/G1 and G2 + M phase cells.

Detailed analysis of the temporal changes in cell‐cycle phase distribution following treatment with 2.5 × 10−8 and 10−7 M 1,25‐(OH)2D3 showed an initial accumulation of cells in Go/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in Go/G1 phase and an increase in G2 + M. In contrast cells treated with 10−7 M 1,25‐(OH)2D3 had significantly elevated % Go/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from Go/G1 following 4 days of exposure to 10−7 M 1,25‐(OH)2D3. This accumulation of cells in Go/G1 was accompanied by a fall in % S phase cells. After 3 days of treatment the % Go/G1 phase cells began to decline, due presumably to a reduced rate of entry into this phase caused by the transition delay through G2 + M which was present at early times but increased rapidly between days 2 and 4 to reach a maximal effect between 4 and 6 days. These studies confirm the 1,25‐(OH)2D3 effect on progression through G1 phase previously reported in hemopoietic cells. Furthermore they document an additional transition delay in G2 + M which is a major contributor to the known growth inhibitory effects of this hormone in T 47D human breast cancer cells. Further study of these effects may lead to a better understanding of hormonal regulation of cellular replication in human breast cancer.
Stimulation of a Mr 80,000 protein phosphorylation by EGF in EGF receptor‐hyperproducing human tumor cells
Authors: Hirai Masamichi , Shimizu Nobuyoshi
Published: February 2005

NA and Ca9‐22 cells derived from squamous cell carcinomas of the tongue possess a large number of epidermal growth factor (EGF) receptors (2.0 × 106 and 1.3 × 106 receptors/cell, respectively). In these cell lines, EGF stimulated receptor autophosphorylation and phosphatidylinositol (PI) turnover. Furthermore, EGF enhanced the phosphorylation of an acidic protein of Mr 80,000. Phosphorylation of this protein was also stimulated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a phorbol ester tumor promoter, and was mainly at serine residues. Phosphopeptide mapping using protease V8 or trypsin indicated that Mr 80,000 proteins isolated from the EGF‐ and TPA‐treated cells were identical. The Mr 80,000 protein was present mainly in the cytosol, but it became closely associated with the membrane as a phosphorylated form upon EGF or TPA stimulation. These results suggest that the EGF‐stimulated phosphorylation of the Mr 80,000 acidic phosphoprotein in EGF receptor‐hyperproducing tumor cells is mediated through the activation of PI turnover and protein kinase C.
Cortisol induction of metallothionein in primary culture of rainbow trout hepatocytes
Authors: Hyllner Sven Johan , Andersson Tommy , Haux Carl , Olsson Per‐Eric
Published: February 2005

In the present experiment, metabolically active primary cultures of rainbow trout hepatocytes were utilized to study the inducibility of metallothionein after cortisol treatment. Glucocorticoid induction of metallothionein has previously only been demonstrated in mammalian systems. We now demonstrate the inducibility of rainbow trout metallothionein by cortisol treatment of primary hepatocytes. A 90% elevation above control levels was achieved within 8 days of treatment. Zinc treatment was performed to evaluate the system, and 100 μM zinc in the culture medium resulted in a 350% increase of the metallothionein levels. We conclude that primary culture of rainbow trout hepatocytes constitutes an efficient system for studies of MT induction kinetics.
Interleukin 1 stimulates prostaglandin synthesis and cyclic AMP accumulation in Swiss 3T3 fibroblasts: Interactions between two second messenger systems
Authors: Burch Ronald M. , White Marsha F. , Connor Jane R.
Published: February 2005

Biochemical events elicited by interleukin 1 (IL–1) were studied in Swiss 3T3 fibroblasts. One hour after its addition, IL–1 stimulated synthesis of prostaglandin E2 (PGE2), which continued to accumulate for 4 days. IL–1 also stimulated cAMP accumulation. Indomethacin blocked cAMP accumulation in response to IL–1, suggesting that PG2 was responsible for the increase. Addition of exogneous PGE2 to indomethacin‐treated cells restored cAMP accumulation. IL–1 enhanced thymidine incorporation, and indomethacin attenuated responses to lower concentrations. Thus, PGE2 appeared to play a role in the ability of low concentrations of IL–1 to stimulate thymidine incorporation. PGE2 augmented thymidine incorporation by increasing cAMP accumulation because in the presence of indomethacin addition of exogenous cAMP enhanced thymidine incorporation in response to low concentrations of IL–1. Elevated cAMP further stimulated PGE2 synthesis. Thus, PGE2 and cAMP interacted to potentiate their mutual accumulation. In summary, IL–1 stimulates PGE2 synthesis. PGE2, in turn, stimulates cAMP accumulation which potentiates IL–1–stimulated PGE2 synthesis and thymidine incorporation.
HeLa cell mutants resistant to epidermal growth factor ricin A‐chain conjugate
Authors: Banker Deborah , Herschman Harvey R.
Published: February 2005

Epidermal growth factor (EGF) was linked to the toxic A chain of ricin toxin (RTA) to produce an EGF‐receptor‐specific cytotoxic agent, EGF‐RTA. Three EGF‐RTA‐resistant mutants of the human HeLa cell line were selected. These mutant cell lines are 10‐fold to more than 100‐fold more resistant to EGF‐RTA when compared to HeLa cells. The EGF‐RTA‐resistant mutants have at least as many EGF receptors as parent cells; the basis for the EGF‐RTA‐resistant phenotype must be distal to EGF binding. The EGF‐RTA‐resistant cells are not cross‐resistant to ricin or to diphtheria toxin; their mutant phenotype appears to be EGF specific. The EGF‐RTA‐resistant mutants are able to internalize and degrade EGF. However, the mutants have altered EGF receptor down‐regulation and phorbol 12‐tetradecanoate 13‐acetate modulation properties. EGF‐RTA/ammonium chloride and EGF‐RTA/adenovirus co‐treatment data suggest that the mutant defect(s) which confers EGF‐RTA resistance is either in the endosome or at a step(s) in the intracellular EGF processing pathway between the endosome and the lysosome.
Ca++‐ionophore‐induced dissipation of intracellular proton gradients in rat thymocytes
Authors: Dell'Antone Paolo
Published: February 2005

Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 μM in about 10 minutes at 37oC. The addition of (NH4)2SO4 (at a final concentration of several mMolars) releases about 30% of the incorporated dye into the medium. The NH4+‐releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 μM of the Ca++‐ionophore A23187. Dye uptake is associated with an absorbance change at 492 mm and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within the acidic cellular compartments. In a Ca++‐containing physiological saline, the addition of A23187 (at a final concentration of 8 μM) at the end of the dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca++, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 μM A23187 in Ca++‐containing saline completely abolished NH4+‐sensitive dye uptake; less A23187 (0.5 μM) and like or a longer incubation period brought about a striking decrease in NH4+‐sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external Ca++, A23187 impaired cell capacity to incorporate dye in a ΔpH‐dependent manner, but a longer incubation time or higher concentrations of the ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca++ or also Mg++)‐H+ exchange.
Evidence for two pathways of protein kinase C induction of 2AR expression: Correlation with mitogenesis
Authors: Smith James H. , Denhardt David T.
Published: February 2005

2ar is a cDNA clone of an mRNA that is inducible by 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA) in mouse JB6 epidermal cells. This mRNA is stably induced in confluent cells but only transiently induced in subconfluent cells; the elevated level of 2ar mRNA in TPA‐treated cells appears to be the result of enhanced transcription (Smith and Denhardt: Journal of Cellular Biochemistry 34:13–22, 1987). Phorbol dibutyrate, teleocidin, and aplysiatoxin, which activate protein kinase C and are also tumor promoters, are shown here to induce 2ar to the same extent as TPA at both cell densities. The increased expression by TPA was prevented by cycloheximide and by H7, a specific inhibitor of protein kinase C. Epidermal growth factor, platelet‐derived growth factor, and the nonphorbol promoter diethylhexylphthalate were more effective inducers in confluent than in subconfluent cultures. All‐trans retinoic acid, dexamethasone and fluocinolone acetonide, inhibitors of tumor promotion, diminished 2ar induction in both confluent and subconfluent cells. In TPA‐treated subconfluent cultures indomethacin produced a slight inhibition, whereas difluoromethyl ornithine potentiated the induction. In TPA‐treated confluent cultures, in contrast, indomethacin enhanced 2ar mRNA levels and difluoromethyl ornithine was inhibitory. We conclude that the protein kinase C‐mediated induction of 2a‐ expression is controlled by different pathways in subconfluent cultures and confluent cultures, indicative of apparent changes in the regulation of gene expression as proliferating cells become quiescent.
Inhibition of heat shock (stress) protein induction by deuterium oxide and glycerol: Additional support for the abnormal protein hypothesis of induction
Authors: Edington Brent V. , Whelan Sandra A. , Hightower Lawrence E.
Published: February 2005

The patterns of radioactively labeled proteins from cultured chicken embryo cells stressed in the presence of either D2O or glycerol were analyzed by using one‐dimensional polyacrylamide gel electrophoresis. These hyperthermic protectors blocked the induction of stress proteins during a 1‐hour heat shock at 44°C. The inhibitory effect of glycerol but not D2O on the induction of heat shock proteins could be overcome by increased temperature. By using transcriptional run‐on assays of isolated nuclei and cDNA probes to detect hsp70‐ and hsp88‐specific RNA transcripts, it was shown that the D2O and glycerol blocks occurred at or before transcriptional activation of the hsp70 and hsp88 genes. After heat‐stressed cells were returned to 37°C and the protectors were removed, heat shock proteins were inducible by a second heating. This result and the fact that the chemical stressor sodium arsenite induced stress proteins in glycerol medium indicated that the treatments did not irreversibly inhibit the induction pathways and that the stress response could be triggered even in the presence of glycerol by a stressor other than heat. In principle then, cells incurring thermal damage during a 1‐hour heat shock at 44°C in D2O or glycerol medium should be competent to respond by inducing heat shock proteins during a subsequent recovery period at 37°C in normal medium. We found that heat shock proteins were not induced in recovering cells, suggesting that glycerol and D2O protected heat‐sensitive targets from thermal damage. Evidence that the heat‐sensitive target(s) is likely to be a protein(s) is summarized. During heat shocks of up to 3 hours duration, neither D2O nor glycerol significantly altered hsp23 gene activity, a constitutively expressed chicken heat shock gene whose RNA transcripts and protein products are induced by stabilization (increased half‐life). During a 2‐hour heat shock, glycerol treatment blocked the heat‐induced stabilization of hsp23 RNA and proteins; however, D2O treatment only blocked RNA transcript stabilization, effectively uncoupling the hsp23 protein stabilization pathway from hsp23 RNA stabilization and transcriptional activation of hsp70 and hsp88 genes.
Elevated c‐fos expression inhibits differentiation of L6 rat myoblasts
Authors: Rahm Magnus , Jin Pei , Sümegi Janos , Sejersen Thomas
Published: February 2005

Expression of c‐fos is induced by a number of signals in several cell systems. Although the exact function of the c‐fos product is unknown, it has been implicated to be of importance for both cell growth and differentiation (Verma and Sassone‐Corsi, 1987). To analyze how c‐fos expression relates to in vitro myogenic differentiation, the kinetics of c‐fos mRNA expression during spontaneous in vitro differentiation of L6J1 myoblasts was examined; c‐fos transcripts were most abundant at day 4 of the differentiation process. Multinucleated myotubes and expression of α‐actin and myosin heavy chain (MHC) mRNA appeared later, at day 6 or 7, and increased to maximal levels after 10 days in culture. To analyze further the relation between c‐fos expression and L6J1 myogenic differentiation, L6J1 myoblasts were transfected with expression vectors containing the murine c‐fos gene driven by a metallothionein promoter. The growth rate of c‐fos‐transfected L6J1 cells did not differ from that of control cells. However, formation of myotubes was significantly reduced in c‐fos‐transfected L6J1 cultures compared with neo‐transfected controls. Myotube formation and expression of the myogenic markers α‐actin and MHC were reduced in subclones expressing high levels of c‐fos, but not in subclones with lower levels of c‐fos expression. These results indicate that a marked elevation of c‐fos expression at least partially inhibits L6J1 myogenic differentiation.
Human microvascular endothelial cells express integrin‐related complexes that mediate adhesion to the extracellular matrix
Authors: Cheng Yao‐Fen , Kramer Randall H.
Published: February 2005

Microvascular endothelial cells (MEC) must use a set of surface receptors to adhere not only to the vascular basement membrane but, during angiogenic stimulation, to the interstitium. We examined how cultured MEC Isolated from human foreskin interact with their subendothelial matrix. MEC were able to attach to diverse extracellular matrix proteins, including fibronectin (Fn), vitronectin (Vn), laminin (Ln), type I and IV collagen, as well as to fibrinogen and gelatin. Adhesion to Fn, but not to laminin or collagens, was specifically blocked in the presence of Arg‐Gly‐Asp (RGD)‐containing peptides. When surface radioiodinated MEC were solubilized and subjected to affinity chromatography on Fn‐Sepharose columns, two polypeptides of 150 and 125 kD, corresponding to the integrin heterodimer α5β1, were identified. MEC also express a complex of 150 (α) and 95 kD (β3) that is related to the Vn receptor. Immunofluorescent staining of MEC cultures with antibodies to the integrin β1 subunit demonstrated receptors on the basolateral surface at focal adhesion plaques that co‐localized with vinculin and with Fn‐positive matrix fibers. Occasionally, antibodies to the Vn receptor stained the vinculin‐positive focaladhesion plaques that frequently co‐localized with the β1 complex. However, in cultures of MEC that were attached to substrates coated with alternating strips of Fn and Vn, the β1 complex was preferentially localized to the Fn substrate, while the Vn receptor was concentrated on the Vn substrate. The results indicate that MEC express at least two different heterodimer adhesion receptors that belong to the integrin superfamily and appear to have distinct ligand specificities: the Fn receptor and the Vn receptor. These receptors mediate cell adhesion to the extracellular matrix and presumably have an important role in hemostasis and neovascularization.
Control of cell division in hepatoma cells by exogenous heparan sulfate proteoglycan
Authors: Fedarko Neal S. , Ishihara Masayuki , Conrad H. Edward
Published: February 2005

The effects of cell surface heparan sulfate proteoglycan (HSPG) prepared from log and confluent monolayers of a rat hepatoma cell line on hepatoma cell growth were studied. When HSPG isolated from confluent cells was added exogenously to log phase cells, it was internalized and free heparan sulfate (HS) chains appeared transiently in the nucleus. Concurrently, the growth of the treated cells was inhibited, but the cells resumed logarithmic growth as the level of nuclear HS fell, and the cells grew to confluence and became contact inhibited. When HSPG prepared from log‐phase hepatoma cells was added exogenously to log phase cells, it was internalized but very little of the internalized HS appeared in the nucleus, and there was no change in the rate of cell growth. However, when the rate of cell growth was reduced by culture of the cells in serum‐ and insulin‐deficient medium, HSPG prepared from log‐phase cells stimulated the growth rate of these slow‐growing cells. The cell cycle dependency of HSPG uptake and growth inhibition was studied in cultures synchronized by a thymidine/aphidicolin double block. When [35SO4]HSPG from confluent cells was added to synchronized cells just as they were released from the second block, a portion of the [35SO4]HSPG was internalized and [35SO4]HS appeared in the nucleus. However, at mitosis the [35SO4]HS disappeared almost completely from all of the cellular pools, and after mitosis, more of the [35SO4]HSPG was taken up and [35SO4]HS reappeared in the nucleus and remained in the nucleus until the cells divided again. When cultures were released from the aphidicolin block, both control and HSPG‐treated cells progressed through the S, the G2, and the M phases of the cell cycle. However, the length of the G1 phase of the cycle was increased in the HSPG‐treated cells. The treated cultures then progressed through the second S, G2, and M phases. Thus, the inhibition of cell division occurred in the G1, phase of the cell cycle, prior to the G1,/S boundary. Addition of the HSPG to the synchronized cultures just after the first mitosis resulted in an immediate arrest of the cell cycle in G1, These results support the earlier suggestion (M. Ishihara, N.S. Fedarko, and H.E. Conrad 1987 J. Biol. Chem. 262:4708–4716; M. Ishihara and H.E. Conrad (1989) J. Cell Physiol., in press) that the HSPG formed by confluent hepatocytes plays a role in the prevention of cell division, whereas the HSPG formed by exponentially growing cells plays a role in the stimulation of cell division. The inhibition of cell growth results from a block in the G1, phase of the cell cycle prior to the G1,/S boundary.
Differentiation of germ cells in seminiferous tubules transplanted to testes of germ cell‐deficient mice of W/Wv and SI/SId genotypes
Authors: Kuroda Hideya , Nakayama Hiroki , Namiki Mikio , Matsumoto Keishi , Nishimune Yoshitake , Kitamura Yukihiko
Published: February 2005

(WB × C57BL/6)F1‐W/W (hereafter, WCB6F1‐SI/SId) mice are sterile due to the deficient spermatogenesis in the testes. The cause of deficient spermatogenesis in WBB6F1‐W/W mice is considered to be a defect in germ cells themselves, whereas that in WCB6F1‐SI/SId mice is considered to be a defect in tissue environment necessary for differentiation of germ cells. Seminiferous tubules isolated from cryptorchid testes of C57BL/6‐+/+ mice were transplanted into the testes of WBB6F1‐W/W and WCB6F1‐SI/SId mice to clarify that the extratubular environment of these mice was intact or not. Type A spermatogonia in the transplanted tubules normally differentiated into spermatids, suggesting that the extratubular environment is intact in both WBB6F1‐W/W and WCB6F1‐SI/SId mice.
Cyclic AMP can partially restore platelet‐derived growth factor‐stimulated prostaglandin E2 biosynthesis, and calcium mobilization in EJ‐ras‐transformed NIH‐3T3 cells
Authors: Olinger P. L. , Benjamin C. W. , Gormacn R. R. , Connor J. A.
Published: February 2005

NIH‐3T3 cells transformed by the EJ‐ras oncogene display reduced platelet‐derived growth factor (PDGF)‐stimulated phospholipase C activity as measured by inositol 1,4,5‐triphosphate (IP3) synthesis and Ca2 + mobilization. The lack of PDGF‐stimulated Ca2+ mobilization in EJ‐ras transformed cells is not due to a loss of IP3 sensitivity, because microinjected IP3 elevates intracellular Ca2+. Treatment of EJ‐ras transformed cells with cholera toxin or 8‐bromo‐cyclic AMP, but not pertussis toxin or the β‐subunit of cholera toxin, results in a slight recovery of PDGF‐stimulated IP3 synthesis, a marked increase in intracellular Ca2+ mobilization, and an almost complete recovery of prostaglandin E2 biosynthesis. These data suggest that EJ p21‐mediated inhibition of PDGF‐stimulated intracellular events can be partially and transiently reversed by cyclic AMP.
Regulation of mitosis onset and thymidine kinase activity during the cell cycle of Physarum polycephalum plasmodia: Effect of fluorodeoxyuridine
Authors: Wright M. , Tollon Y.
Published: February 2005

The effects of fluorodeoxyuridine were investigated during three events of the cell cycle: S‐phase, mitosis, and the cyclic synthesis of thymidine kinase in the synchronous plasmodium of the myxomycete Physarum. DNA synthesis was inhibited, and there was limited action on other macromolecular syntheses. When DNA synthesis was slowed down, onset of the following increase of thymidine kinase synthesis occurred at approximately the same time as in the control, but mitosis was blocked in a very early prophase stage and metaphase was never observed. These effects were suppressed when the action of fluorode‐oxyuridine was prevented by the addition of thymidine to the medium. In agreement with the action of aphidicolin and hydroxyurea, these observations show that: (1) perturbation of the S‐phase does not prevent the nuclei from entering a very early prophase stage, but it does prevent them from proceeding through metaphase; (2) blockage of DNA synthesis does not perturb the normal timing of the triggering of thymidine kinase synthesis; and (3) the signal that triggers the arrest of thymidine kinase synthesis is postmitotic and does not require extensive DNA synthesis. In contrast with hydroxyurea and aphidicolin, in the presence of fluorodeoxyuridine metaphase was not observed. Thus, the triggering of thymidine kinase synthesis is unambiguously dissociated from metaphase and postmitotic events. Because synthesis of thymidine kinase remains under the control of temperature shifts from 22 to 32°C, a simple model of the cell cycle involving two regulatory pathways could account for the triggering of thymidine kinase synthesis, early prophase stage, and metaphase.
Kinetics of α2‐macroglobulin endocytosis and degradation in mutant and wild‐type Chinese hamster ovary cells
Authors: Yamashiro Darrell J. , Borden Laurence A. , Maxfield Frederick R.
Published: February 2005

The production of Chinese hamster ovary (CHO) cell mutants which are defective in endocytosis has led to a greater understanding of the process by which cells sort ligands and their receptors. Robbins and coworkers have obtained CHO mutants which are resistant to diphtheria toxin, defective in the delivery of endocytosed lysosomal enzymes to lysosomes, and have a decreased uptake of iron from transferrin (Robbins et al.: 1. Cell Biol. 96:1064–1071, 1983). We have previously shown that these CHO mutants are markedly deficient in the acidification of early endocytic compartments (Yamashiro and Maxfield: J. Cell Biol. 105:2713–2721, 1987). In this study we examined the endocytosis of α2‐macroglobulin (α2M) to determine whether the defects in early endosome acidification would alter the processing of this ligand. We found that the CHO mutants DTG 1–5–4 and DTF 1–5–1 bind, internalize, and degrade 125l‐α2M in a manner similar to the wild‐type cells. We also found that the CHO mutants retain the ability to recycle the receptors for α2M. Since the binding of α2M is greatly reduced at mildly acidic pH (≈︁6.8), only slight acidification of the endosomal compartment should be sufficient to achieve sorting of a2M from its receptor. In contrast, lysosomal enzymes require more acidic conditions (pH < 6.0) for dissociation. The different behavior of the two ligands provides biochemical evidence for a partial (but not complete) defect in early endosome acidification in the mutants. The data also indicate that pH regulation in a relatively narrow range can achieve differential sorting of various ligands.
Murine B‐cell stimulatory factor‐1 (BSF‐1)/Interleukin‐4 (IL‐4) is a multilineage colony‐stimulating factor that acts directly on primitive hemopoietic progenitors
Authors: Kishi Kenji , Ihle James N. , Urdal David L. , Ogawa Makio
Published: February 2005

Interleukin‐4 (IL‐4), which was originally identified as a B‐cell growth factor, has been shown to produce diverse effects on hemopoietic progenitors. The present study investigated the effects of purified recombinant murine IL‐4 on early hemopoetic progenitors in methylcellulose culture. IL‐4 supported the formation of blast cell colonies and small granulocyte/macrophage (GM) colonies in cultures of marrow and spleen cells of normal mice as well as spleen cells of mice treated with 150 mg/kg 5‐fluorouracil (5‐FU) 4 days earlier. When the blast cell colonies were individually picked and replated in cultures containing WEHI‐3 conditioned medium and erythropoietin (Ep), a variety of colonies were seen, including mixed erythroid colonies, indicating the multipotent nature of the blast cell colonies supported by IL‐4. To test whether or not IL‐4 affects multipotent progenitors directly, we replated pooled blast cells in cultures under varying conditions. In the presence of Ep, both IL‐3 and IL‐4 supported a similar number of granulocyte/erythrocyte/macrophage/megakaryocyte (GEMM) colonies. However, the number of GM colonies supported by IL‐4 was significantly smaller than that supported by IL‐3. When colony‐supporting abilities of IL‐4 and IL‐3 were compared using day‐4 post‐5‐FU spleen and day‐2 post‐5‐FU marrow cells, IL‐4 supported the formation of fewer blast cell colonies than did IL‐3. IL‐4 and IL‐6 revealed synergy in support of colony formation from day 2 post‐5‐FU marrow cells. These results indicate that murine IL‐4 is another direct‐acting multilineage colonystimulating factor (multi‐CSF), similar to IL‐3, that acts on primitive hemopoietic progenitors.
Role of mevalonic acid in the regulation of natural killer cell cytotoxicity
Authors: Cutts J. Lynn , Scallen Terrence J. , Watson John , Bankhurst Arthur D.
Published: February 2005

Considerable evidence has accumulated for a role of a nonsteroidal mevalonate product in the regulation of DNA replication and cell division. We report here a similar requirement for mevalonate in a nonreplicative function, that of natural killer (NK) cell cytotoxicity. Treatment of NK cells with 10 μM compactin for 48 hr results in a significant inhibition of cytotoxicity which can be completely reversed by treatment with 1 mM mevalonate, but not cholesterol, dolichol, or isopentenyl adenine. Protein and RNA synthesis appear to be involved in this reversal. Treatment with compactin and reversal with mevalonate do not affect the phenotypic distribution of the effector cell population, and the cell type involved in the inhibition and reversal of cytotoxicity is a CD16 (Leu 11)‐, Leu 19‐positive, large granular lymphocyte. The conjugation of the target and effector cell early in the lytic pathway is inhibited by compactin treatment of the effector cell population, and this inhibition is reversed by mevalonate.
Injury‐induced release of basic fibroblast growth factor from bovine aortic endothelium
Authors: Gajdusek Corinne M. , Carbon Sheila
Published: February 2005

Although the basic fibroblast growth factor (bFGF) gene lacks a traditional consensus signal peptide domain indicative for secretion, many cell types have receptors for bFGF. Since endothelium is a rich source of cell‐associated bFGF, we asked under what conditions could bFGF be released or secreted from confluent cultures of bovine aortic endothelial (BAE) cells. The level of bFGF in BAE cell lysates was compared with the level of heparin‐releasable bFGF in intact BAE cell monolayers, intact cells with exposed extracellular matrix (nonlytic matrices), and extracellular matrices prepared by cell lysis (lytic matrices). Less than 10% of total cell‐associated bFGF was released from intact cell monolayers and nonlytic matrices. In contrast, the levels of bFGF released from lytic matrices depended upon the conditions used to prepare the matrices. Cell lysis at neutral pH generated matrices that released the highest bFGF levels (approximately 50% of total cell‐associated bFGF). These matrices were heavily contaminated by histones, indicating the cellular release and adsorption of intracellular proteins to the matrix. Matrices prepared by BAE cell exposure to basic pH (100 mM NH4OH) contained low bFGF content and minor histone contamination. These latter matrices were chosen to study bFGF sequestration, under physiological conditions, into the extracellular matrix of confluent BAE cell cultures. Incubation with endotoxin, an agent acutely toxic to BAE cells, resulted in cellular release and adsorption of endogenous bFGF to cells and matrices, accompanied by histone deposition in the matrices. These results suggested that one mechanism for bFGF release from BAE cell monolayers was passive release induced by severe cell injury and/or cell lysis with secondary adsorption to the matrix.
Relationship between changes in ploidy and stable cellular resistance to hydrogen peroxide
Authors: Spitz Douglas R. , Mackey Michael A. , Li Gloria C. , Elwell James H. , McCormick Michael L. , Oberley Larry W.
Published: February 2005

Stable hydrogen peroxide (H2O2)‐resistant variants of the Chinese hamster ovary HA‐1 line have been isolated by culturing cells in progressively increasing concentrations of H2O2 (>200 days, in 50–800 μM H2O2). Increases in catalase activity in these variant cell lines were shown to correlate with increased H2O2 resistance. Stable (>240 days) H2O2‐resistant cell lines, seven quasidiploid (21–22 chromosomes/cell) and six quasitetraploid (40–44 chromosomes/cell) were clonally isolated from the 800 μM adapted H2O2‐resistant variants which were heterogeneous with respect to ploidy. The H2O2 dose‐modifying factors (DMFs) were 3, 5, 8, 13, 15, 26, and 27 for the seven quasidiploid cell lines, and 21, 32, 38, 40, 42, and 49 for the six quasitetraploid cell lines. The mean DMF was 14±10 for the former and 37±10 for the latter. Our data show that on the average the quasitetraploid cell lines were significantly more resistant to H2O2‐mediated cell killing than the quasidiploid cell lines derived from the same mixed population of 800 μM H2O2‐adapted cells. When catalase activities (k units/cell) of the HA‐1 cells and three of the clonally derived cell lines (two quasidiploid and one quasitetraploid) were determined and plotted vs. H2O2–DMF, a positive linear correlation was obtained (correlation coefficient = 0.99). This result was further confirmed when immunoreactive catalase protein/cell was detected by Western blots. Our data show that chronic exposure of cells to H2O2 stress (800 μM) was accompanied by increases in quasitetraploid cells within the population. Quasitetraploid cell lines derived from this population demonstrated increased stable H2O2‐resistance which may be related to stable increases in the expression of catalase.
Establishment and partial characterization of SV40 virus‐immortalized hepatocyte lines of normal and lethal mutant mice carrying a deletion on chromosome 7
Authors: Paul Dieter , Kwon Byoung S. , Höhne Martin , Tönjes Ralf , Haq Asifa K. , Hoffmann Birgit
Published: February 2005

Deletions in chromosome 7 of the mouse have been shown to cause failure of expression of various hepatocyte‐specific genes in newborn deletion homozygotes, including the gene encoding tyrosine amino transferase (TAT) (EC 2.6.1.5) (Gluecksohn‐Waelsch, 1979). Primary liver cultures of newborn albino deletion mutant mice (C14CoS/C14CoS) and of phenotypically normal mice (C14CoS/Cch or Cch/Cch) were infected with SV40 virus and multiplying hepatocytes selected in arginine‐deficient medium containing epidermal growth factor (EGF), insulin, and hydrocortisone (HC). Resulting normal (NMH‐ch) and mutant (NMH‐m14) hepatocyte lines expressing integrated viral transforming sequences did not senesce, they multiplied autonomously of EGF in medium with insulin plus HC, and they retained hepatocyte‐specific functions. Both lines synthesized arginine and contained albumin and alpha‐fetoprotein (AFP) mRNAs. TAT‐specific mRNA was detected in normal but not in mutant hepatocyte lines. A fragment of the mouse tyrosinase gene, known to map at the albino locus (c) within the region deleted in the C14CoS mutant, hybridized with a 2.5 kb EcoRI fragment of normal NMH‐ch DNA, whereas this fragment was undetectable in mutant NMH‐m14 DNA. These immortalized hepatocyte lines reflect important properties of normal and mutant liver tissues from which they were derived. The deletion mutant mouse cell lines may be useful for complementation studies involving sequences corresponding to the deletions that encode regulatory gene(s) involved in the control of inducible expression of certain hepatocyte‐specific genes such as TAT.
Will heat‐stable proteins nonspecifically protect cells from thermal stress?
Authors: Prenninger Siegfried W. , Li Gloria C.
Published: February 2005

In cell‐free systems, stress‐resistant proteins nonspecifically stabilize stress‐susceptible proteins. This mechanism has been suggested to contribute to thermotolerance in cells (Minton et al.: Proc. Natl. Acad. Sci. USA, 79:7107–7117,1982). To test this hypothesis, red‐blood‐cell‐mediated microinjection was used to transfer macromolecules into monolayers of CHO cells. We introduced the heat‐stable proteins fetuin and ovomucoid into RBCs during hypotonic hemolysis and then fused the RBCs to CHO cells with polyethylene glycol as fusogen. Fetuin and ovomucoid were successfully transferred into 36–55% of the CHO cells as demonstrated by fluorescence of FITC‐conjugated proteins. The plating efficiency of these CHO cells after fusion ranged from 35% to 60%. Three hours after fusion, CHO cells microinjected with fetuin or ovomucoid were exposed to 43°C for 0–180 min or 45°C for 0–40 min, and thermal survival was determined. There was no difference in cell survival between control untreated cells, control cells fused with nonloaded RBCs, and cells fused with RBCs loaded with fetuin or ovomucoid.

While our results do not support the hypothesis that heat‐stable proteins nonspecifically protect cells from thermal stress, several posslble explanations are provided for this observation.
Biosynthesis of proteoglycans by proliferating and differentiating normal human keratinocytes cultured in serum‐free medium
Authors: Rahemtulla Firoz , Moorer Candace M. , Wille John J.
Published: February 2005

Normal human keratinocytes (NHK) were cultured in serum‐free medium, containing low (0.1 mM) or high (2 mM) calcium, to obtain proliferating and differentiating cultures, respectively. Proteoglycan (PG) synthesis of proliferating and differentiating NHK was investigated. Cultures were labeled with 35S‐sulfate, and the PGs were extracted from medium and cell layer. The newly synthesized PGs were isolated by ion‐exchange chromatography on a column of DEAE‐Sephacel. The molecular properties of the PGs and the size and composition of glycosami‐noglycans (GAGs) were determined. In general, the PGs are of relatively small size (Mr 70,000‐120,000). The PGs of proliferating cultures are larger in molecular size than the PGs of differentiating cultures, and this is due to the degradation of the GAG chains. The molecular weight of the GAG chains of proliferating NHK ranged from 4,800 to 22,000, and the range for GAGs from differentiating cultures varied from 2,800 to 9,600. By compositional analysis, these PGs proved to contain heparan sulfate, chondroitin sulfate, and dermatan sulfate as determined by nitrous acid degradation, and chondroitinase ACII and ABC digestion. No significant differences were found in the overall GAG composition of the medium secreted PGs of proliferating and differentiating cultures. In contrast, cell‐associated PGs of differentiating cells had higher levels of heparan sulfate than those of proliferating cells.
Characterization of the receptor for insulin‐like growth factor II in bone cells
Authors: Mohan Subburaman , Linkhart Thomas , Rosenfeld Ron , Baylink David
Published: February 2005

We have previously shown that insulin‐like growth factor II (IGF‐II) is produced by bone cells and that IGF‐II stimulates cell proliferation and collagen synthesis in bone cells. We now extend these in vitro findings by demonstrating specific IGF‐II binding to bone cells derived from newborn mouse calvaria and embryonic chick calvaria. The kinetics of [125]I IGF‐II binding in embryonic chick calvaria cells showed time and temperature dependence. Scatchard analysis of [125]I IGF‐II binding to chick calvaria cells showed an apparent Kd of 1.4 × 1010 M, with a calculated receptor site concentration of 40,000/cell. The specificity characteristics showed that IGF‐II was significantly more potent than IGF‐I or insulin in displacing IGF‐II tracer. Competition for binding of [125]I IGF‐II by unlabeled IGF‐II showed a dose‐dependent displacement between 0.5 and 25 ng/ml. Fifty percent displacement of [125]I IGF‐II binding to chick and mouse calvarial cells was achieved at 1‐2 ng/ml; 90% of specific binding of [125]I IGF‐II was displaceable in the presence of 125 ng/ml of unlabeled IGF‐II. IGF‐I showed less than 5% cross reactivity for displacement of [125]I IGF‐II binding to chick and mouse bone cells. Type II receptor inhibitory antibodies, R‐II‐PAB1 inhibited the binding of [125]I IGF‐II to mouse bone cells and H‐35 rat hepatoma cells (which contain type II but not type I receptors) in a dose‐dependent manner. R‐II‐PAB1 also inhibited basal cell proliferation as well as IGF‐II‐, IGF‐I‐, and fibroblast growth factor (FGF)‐induced cell proliferation in mouse bone cells. In chick calvaria bone cells and TE89 human osteosarcoma cells, R‐II‐PAB1 inhibited neither binding of [125]I IGF‐II nor IGF‐ll‐induced cell proliferation. These results together with our findings that IGF‐II increased chick bone cell proliferation in the presence of maximal doses of IGF‐I suggest that at least part of the mitogenic action of IGF‐II is mediated through type II rather than type I receptors in bone cells.
Reduction of adenine nucleotide content of clone 4 mdck cells: Effects on multiplication, protein synthesis, and morphology
Authors: Ladino C. , Schneeberger E. E. , Rabito C. A. , Lynch R. D.
Published: February 2005

The antitumor agent hadacidin (N‐formyl‐hydroxyamino‐acetic acid), at 4 mM, inhibited the multiplication of clone 4 Madin Darby canine kidney (MDCK) cells within 24 hr. Growth resumed rapidly upon replacement of hadacidin with aspartate, an observation consistent with the drug's action as a competitive inhibitor of adenylosuccinate synthetase, an enzyme in adenine nucleotide biosynthesis. Data indicate that the drug‐treated cells were arrested in S phase of the cell cycle. Accompanying inhibition of multiplication was a 16‐fold increase in the area occupied by the cells and a refractoriness to release by treatment with trypsin. None of these changes occurred when 0.5 mM adenosine was included in the incubation mixture containing the inhibitor. Hadacidin decreased the adenosine triphosphate (ATP) and cyclic adenosine monophosphate (cAMP) content of the cells as well as the rate at which 3H‐leucine was incorporated into protein. In the presence of 1 mM dibutyryl cAMP and theophylline, the drug had no effect on cell division and protein synthesis. The data suggest that, in clone 4 MDCK cells, the effects of hadacidin are mediated by diminishing the level of cAMP.
Coupling of bradykinin receptors to phospholipase C in cultured fibroblasts is mediated by a G‐protein
Authors: Etscheid Beth G. , Villereal Mitchel L.
Published: February 2005

In cultured foreskin fibroblasts, bradykinin stimulates inositol phosphate generation, arachidonic acid release, and Na+/H+ exchange, with doses of 1‐3 nM yielding half‐maximal stimulation. Binding of 3H‐bradykinin to these cells demonstrates a single receptor site with a Kd of 2.0 nM and a Bmax of 91 fmoles/mg protein. Bradykinin analogs of the B2 type inhibit this binding. GTP synergizes with bradykinin to stimulate phosphatidylinositol turnover in permeabilized fibroblasts and GTP‐γ‐S decreases the Bmax of bradykinin binding to fibroblast membranes, indicating that a G‐protein couples the receptor to phospholipase C. Pretreatment of fibroblasts with either cholera or pertussis toxin enhances bradykinin stimulation of inositol phosphate accumulation.
Volume changes of an endothelial cell monolayer on exposure to anisotonic media
Authors: Mazzoni Michelle C. , Lundgren Evy , Arfors Karl‐E. , Intaglietta Marcos
Published: February 2005

The osmotic process plays an important role in controlling the distribution of water across cell membranes and thus the cell volume. A system was designed to detect the volume changes of an endothelial cell monolayer when cells were exposed to media with altered osmolalities. Electrodes housed in a flow chamber measured the resistance of ionic media flowing over a cultured cell layer. Assuming the cell membrane acts as an electrical insulator, volume changes of the cell layer can be calculated from the corresponding changes in chamber resistance. The media used in the experiments had osmolalities in the range 120–630 mmol/kg. When cells were exposed to hypertonic media, there was rapid shrinkage with an approximate 30% reduction in total cell volume for a twofold increase in osmolality. On exposure to hypotonic media, the cells initially swelled with an approximate 20% volume increase for a decrease in osmolality by half. With sustained exposure to low osmolality media, there was a gradual and partial return of cell volume towards isotonic values that started 10 minutes after and was complete within 30 minutes of the osmolality alteration. This finding suggests regulatory volume decrease (RVD); however, no regulatory volume increase (RVI) was observed with the continued exposure to hypertonic media over 45 minutes.