bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of proliferation of bovine aortic endothelial cells, smooth muscle cells, and adventitial fibroblasts in collagen lattices
Authors: Weinberg Crispin B. , Bell Eugene
Published: February 2005

We compared the proliferation of bovine aortic cells grown in collagen lattices. Smooth muscle cells continued to divide for 2 weeks while adventitial fibroblasts ceased to divide after 4–5 days. Endothelial cells did not proliferate within an untreated collagen lattice; however, if the lattice was covered with culture medium, endothelial cells populated its surface and proliferated to form a monolayer. We also found that both smooth muscle cells and endothelial cells, like fibroblasts, are able to contract a collagen lattice to a small fraction of its original volume, although endothelial cells are able to do so only if the lattice is covered with culture medium.
Calcium regulation of phosphatidyl inositol turnover in macrophage activation by formyl peptides
Authors: Holian Andrij , Stickle Douglas F.
Published: February 2005

Stimulation by the tripeptide N‐formyl norleucyl leucyl phenylalanine (FNLLP) of the guinea pig alveolar macrophage gives rise to transient production of superoxide anion (O2−). Components of the phosphatidyl inositol (PI) cycle (phosphatidic acid) (PA), phosphatidyl inositol‐4,5‐bisphosphate (TPI) and phosphatidyl inositol‐4‐phosphate (DPI) were monitored using 32P in order to examine the possible association of this cycle with the FNLLP‐stimulated production of O2−. Macrophage stimulation by FNLLP led to an increased flux of metabolites through the PI cycle. The level of 32P label in both TPI and DPI rapidly decreased upon exposure to FNLLP, followed by a 5‐min period during which the 32P label in TPI and DPI approached prestimulated levels. During this period, there was a fivefold increase in 32P‐PA. It is suggested that diacylglycerol (DAG) is the O2− production. The importance of continued cycling of PI in the stimulated mechanism is demonstrated by the inhibition by LiCl of the extent, but not the initial rate, of both O2− production and the formation of 32P‐PA upon peptide stimulation after 1‐h preincuexamined. It has previously been demonstrated that intracellular availability of calcium can influence the rate and extent of O2− production. In cells preloaded with quin‐2, which acts as a high‐affinity sink for calcium in the cytosol, the initial rate of FNLLP‐stimulated O2− production is inhibited in low (10 μM) extracellular calcium medium. High extracellular calcium (1 mM) completely reverses this inhibition and also significantly extends the time course of O2− production in both quin‐2 and control cells (Stickle et al., 1984). In parallel with these effects on O2− production, varying calcium conditions is demonstrated to influence the rate and extent of PA formation. These same calcium conditions were found to have little or no effect on the initial unstimulated levels of TPI, DPI, and PA. These results indicate that the influence of an intracellular pool of calcium on O2− production may be via its influence on stimulated PI turnover.
The effect of ionophores and related agents on the induction of doming in a rat mammary epithelial cell line
Authors: Paterson Frieda C. , Graham John M. , Rudland Philip S.
Published: February 2005

Addition of dimethyl sulfoxide (DMSO) and the mammotropic hormones prolactin, hydrocortisone, insulin, and estradiol to confluent cultures of the epithelial cell line Rat Mammary (Rama) 25 increases dramatically the formation of domes in the cell monolayer after 48–72 hr. Associated with the increase in doming is an increase of 24% in the activity of the Na+/K+ ATPase. Both Ca2+ (A23187) and Na+ (monensin, gramicidin J, melittin) ionophores can replace DMSO in inducing domes whilst the K+ ionophore valinomycin inhibits doming. However, there are no synergistic nor additive effects, respectively, with suboptimal or optimal concentrations of A23187 and melittin together. Ouabain, at concentrations which inhibit the Na/K ATPase in vitro, and amiloride, at concentrations reported to inhibit the passive transport of Na+, both inhibit completely the formation of domes induced by DMSO, A23187, and melittin. EGTA, however, inhibits only the induction of doming by DMSO and A23187; it is without effect with melittin. A23187 and melittin induce the major polypeptide changes that occur in doming cultures with DMSO, and most of these changes are also inhibited with ouabain. It is suggested that one possible interpretation of the findings is that the induction of doming by DMSO in Rama 25 cells occurs by means of sequential increases in Ca2+ and Na+ influxes into the cell, and that the increased intracellular concentration of Na+ so produced stimulates the Na+/K+ ATPase, with a net effect of pumping liquid beneath the cellular monolayer.
Differential effects of abrin on normal and tumor cells
Authors: Chan Lee‐Nien Lillian , Li Jiann‐Shyong , Liu Su‐Yin
Published: February 2005

The effects of the plant toxin abrin on normal mouse embryonic fibroblasts (MEF), an untransformed mouse cell line (NIH 3T3), and two mouse tumor cell lines (LMTK− and S‐180) were studied. Measurements of cell growth and colony formation showed that MEF and S‐180 cells were more sensitive to abrin intoxication than NIH 3T3 and LMTK− cells. Also, the effects of abrin on the inhibition of [3H]leucine and [3H]thymidine incorporation were more evident in MEF and S‐180 cells. The basis for these varying responses to abrin by the four different cells was examined. The number of abrin binding sites per cell was determined from [125]abrin binding studies: NIH 3T3 and LMTK− cells had significantly fewer abrin binding sites than MEF and S‐180 cells. The fate of the [125l]abrin after internalization was examined by gel electrophoresis and autoradiography. A pattern of time‐dependent degradation was observed, degradation being more rapid in NIH 3T3 and S‐180 cells than in LMTK− and MEF cells. We conclude that the varying responses of different cells to the toxin abrin may be due to several factors, including the relative number of abrin binding sites on the cell surface and the rate of degradation of the toxin once internalized. The results also show that the sensitivities of the cells to abrin do not necessarily correlate with their normal or neoplastic state.
Phenotypic transformation of normal rat kidney cells in a growth‐factor‐defined medium: Induction by a neuroblastoma‐derived transforming growth factor independently of the EGF receptor
Authors: van Zoellen Everardus J. J. , van Oostwaard Theodora M. J. , van der Saag Paul T. , de Laat Siegfried W.
Published: February 2005

Polypeptide growth factor activity in serum can be destroyed by treatment with dithiothreitol. When such growth‐factor‐inactivated serum is used as a supplement of culture media instead of regular serum, normal rat kidney (NRK) cells become quiescent unless defined polypeptide growth factors like insulin and epidermal growth factor (EGF) are added. On this basis a growth‐factor‐defined medium has been developed for NRK cells, which permits cell proliferation as rapidly as in media supplemented with serum, even at low cell densities. Moreover, cells can be serially passaged in this medium. NRK cells can be induced to grow in semisolid media when incubated with transforming growth factors. The growth‐factor‐defined medium permits soft agar growth experiments of NRK cells, without interference from polypeptide growth factors in serum. Using this assay system we have shown that EGF alone is unable to induce any degree of anchorage‐independent growth in NRK cells. However, a recently identified transforming growth factor from mouse neuroblastoma cells which does not compete with EGF for receptor binding is able to induce progressively growing colonies of NRK cells in soft agar, even without additional EGF.
Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters
Authors: Boonstra Johannes , Mummery Christine L. , van der Saag Paul T. , de Laat Siegrfried W.
Published: February 2005

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high‐affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low‐affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37°C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37°C, the binding capacities of both classes decreased. Furthermore the high‐affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor‐promoting phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). Both compounds caused a decrease of the affinity of the high‐affinity class without affecting the low‐affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low‐affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.
Enterocytic differentiation and glucose utilization in the human colon tumor cell line Caco‐2: Modulation by forskolin
Authors: Rousset Monique , Laburthe Marc , Pinto Moïse , Chevalier Guillemette , Rouyer‐Fessard Christiane , Dussaulx Elisabeth , Trugnan Germain , Boige Nathalie , Brun Jean‐Louis , Zweibaum Alain
Published: February 2005

The human colon cancer line Caco‐2 exhibits after confluency a concomitant increase of glycogen accumulation and an enterocytic differentiation. The purpose of this work was to investigate whether forskolin (FK), an activator of adenylate cyclase, would induce a permanent glycogenolysis and, if so, whether it would result in modifications of the differentiation pattern of the cells. FK activates adenylate cyclase in Caco‐2 cells with an ED50 of 7 × 10−6M. Three different treatment protocols with FK (10−5M) were applied: (1) the cells were treated during all the time in culture (20 days); (2) the treatment was started after confluency; (3) the treatment was interrupted after confluency. The presence of FK results in a permanent stimulation of cAMP accumulation (10 to 20 fold the basal values) and in a permanently reduced glycogen content (30 to 50% of the control values). The rates of glucose consumption are increased three and five fold in protocols 1 and 3 respectively. These metabolic changes are associated with morphological changes (tightening of the intercellular spaces and shortening of the brush border microvilli) and with a dual inhibition of the activities of brush border hydrolases: (a) an inhibition of the post‐confluent increase of activity of sucrase, aminopeptidase N and alkaline phosphatase in the brush border enriched fraction; (b) an inhibition of the post‐confluent increase of activity of sucrase in the cell homogenate. A comparison of the results obtained in each protocol shows that the morphological modifications and the decrease of the enzyme activities in the brush border fraction are regularly associated with an increased cAMP accumulation, whereas the inhibition of the differentiation of sucrase is a direct consequence of the increase in glucose consumption and decrease in glycogen stores.
Carbohydrate metabolism in transforming lymphocytes from the aged
Authors: Tollefsbol Trygve O. , Cohen Harvey J.
Published: February 2005

There is an age‐related decline in immune capacity which has been linked to a decreased response of lymphocytes to mitogens in vitro. During transformation, lymphocytes require a marked increase in energy production and biosynthesis which is supplied primarily by glycolysis. In the elderly, the glycolytic enzymes increase significantly in transforming lymphocytes at least 24 hr later than in the young and then at significantly reduced levels. Glucose utilization is also impaired in stimulated lymphocytes from the elderly but follows the impairment of glycolysis. In stimulated cells from the young, increases in glycolytic enzyme activity levels accompany sharp increases in blastogenesis while a delayed increase in glycolytic enzyme activity in the elderly is accompanied by a delay in blastogenesis. Maximal glycolytic enzyme activity levels are significantly reduced in transformed lymphocytes from the elderly though the number of transformed cells is also significantly reduced. However, glycolytic enzyme activity levels are significantly lower in the elderly than in the young even on a per transformed cell basis. Thus, this reduction cannot be attributed to the lower number of transformed cells that are present in the elderly. This defect in the increase of glycolysis in stimulated cells from the elders suggests an intracellular mechanism which could be related to the impaired lymphocyte stimulation in vitro in the aged.
Diacylglycerol treatment rapidly decreases the affinity of the epidermal growth factor receptors of Swiss 3T3 cells
Authors: Sinnett‐Smith James W. , Rozengurt Enrique
Published: February 2005

The synthetic diacylglycerol 1‐oleoyl‐2‐acetyl glycerol (OAG) and phorbol esters activate protein kinase C in intact cells. We report here that OAG inhibits the binding of 125I‐labelled epidermal growth factor (125I‐EGF) to Swiss 3T3 cells. The inhibition was detected as early as 1 min after treatment at 37°C and persisted for at least 120 min. The effect of OAG was reversed upon removal of this diacylglycerol. Detailed Scatchard analysis of 125I‐EGF binding to Swiss 3T3 cells at 4°C after a 1 h incubation with a saturating dose of OAG at 37°C, demonstrates that this OAG pretreatment does not change the apparent number of EGF receptors but causes a marked decrease in their apparent affinity for the ligand. Prolonged treatment (40 h) of the cells with phorbol dibutyrate (PBt2) which causes a marked decrease in the number of phorbol ester binding sites and in the activity of protein kinase C, prevented the inhibition of 125I‐EGF binding by both PBt2 and OAG. The results support the possibility that protein kinase C plays a role in the transmodulation of the EGF receptor in intact cells.
Reduction of DNA synthesis in aging but still proliferating cells
Authors: Collins James M. , Chu Annie K.
Published: February 2005

It is well known that cell proliferation (and hence, DNA synthesis) declines in human diploid fibroblast‐like cells with increasing passage number. It is not clear whether DNA synthesis declines in the remaining cells that are still actively proliferating. Estimations of cell kinetic parameters permitted extrapolations to be made that reflected the declining numbers of cells still capable of DNA replication. DNA synthesis declined with culture age in intact cells, permeabilized cells, and in the isolated nuclear matrix even when corrected for declining numbers of proliferating cells. With age, DNA polymerase alpha and beta activity in cell lysates declined, but when corrected for the remaining proliferating cells, only polymerase alpha activity declined; DNA polymerase alpha and beta activity bound to the nuclear matrix declined, but when corrected for declining proliferation, no decline was apparent for either enzyme. There was an increase in the number of S1‐nuclease sensitive sites and breaks in the parental DNA of the dividing cells in older cultures. It is suggested that in aging cultures, not only does overall DNA synthesis decline owing to decreasing cell proliferation, but also that DNA synthesis declines in the remaining proliferating cells, that this decline is not due to decreasing amounts of DNA polymerase bound to the nuclear matrix, and that alterations in DNA structure occur.
The metabolism of cobalamin bound to transcobalamin II and to glycoproteins that bind Cbl in HepG2 cells (human hepatoma)
Authors: Hall Charles A. , Green‐Colligan Pamela D. , Begley James A.
Published: February 2005

The binding, internalization, processing and release of labeled cyanocobalamin (CN[57Co]Cbl) bound to human transcobalamin II (TC II) were studied in HepG2 cells, a line of hepatocytes derived from a human hepatoma. The cells bound the TC II‐Cbl by specific, high affinity receptors. Within the cell, the CN‐Cbl was promptly freed from TC II and the CN‐Cbl converted to more active forms including adenosyl Cbl (AdoCbl) and methyl Cbl (MeCbl). Whereas free labeled Cbl was still present at 72 hours after entry, the cells also bound Cbl to an intracellular binder (ICB) presumed to represent the holo enzymes dependent on Cbl. At levels of TC II that saturated the receptors for TC II‐Cbl, much of the Cbl entering the cells remained free and was converted to AdoCbl. Under these circumstances the cells released free Cbl, mostly AdoCbl.

Human R type binders of Cbl, which are glycoproteins and some having a terminal galactose, were bound by the HepG2 cells. The binding was characteristic of the receptor system responsive to a terminal galactose, or asialoglycoproteins, but was inconsistent and of low affinity. Cbl bound to R binder was internalized and converted to coenzyme forms of Cbl, but the process was much less effective than when the Cbl entered via the TC II receptor system. It was concluded that the receptors for R‐Cbl were unlikely to contribute to the physiologic transport of Cbl in man, but may function in some yet unknown way.
A reduction in the in situ rates of oxygen and glucose consumption of cells in EMT6/Ro spheroids during growth
Authors: Freyer J. P. , Sutherland R. M.
Published: February 2005

The rates of consumption of oxygen and glucose by EMT6/Ro cells in multi‐cellular spheroids were measured at various times during normal growth. In situ spheroid cellular consumption rates were similar to those of exponentially growing single cells up to a spheroid diameter of 150 μm. Further growth resulted in decreases in the rates of both oxygen and glucose consumption which were correlated with the increase in spheroid diameter and cell number. At a diameter of 1300 μm, both rates of cellular consumption had decreased by a factor of 2.5. The rates of consumption per unit of nonnecrotic spheroid volume decreased in a similar manner. Measurements with single cells demonstrated that the rate of oxygen consumption was coupled with glucose concentration, and vice versa. The rates of consumption for cells dissociated from small spheroids indicated that there was some effect of the spheroid environment. As the spheroids grew, however, association in the spheroid structure accounted for a smaller proportion of the total observed reduction in the rates of nutrient consumption. The presence of central necrosis also appeared to have no effect on the rates of consumption of these nutrients. Spheroid‐derived cells showed a decrease in cell volume with growth as the cells accumulated in a quiescent state. Measurements with single cells demonstrated that oxygen and glucose consumption were correlated with cell volume and with the development of nonproliferating cells. We conclude that the observed decrease in oxygen and glucose consumption with growth in spheroids is largely due to the progressive accumulation of cells in a quiescent state characterized by an inherently lower cellular rate of nutrient utilization.
Continuous growth and phosphatidylcholine synthesis of rat hepatoma cells in choline‐deprived chemically defined medium
Authors: Yamamoto Katsuhiko , Niwa Akira , Yasumura Yosihiro
Published: February 2005

A choline non‐required cell line was established from a rat hepatoma cell line. The line designated R‐Y121B·cho was able to grow in choline‐deprived medium without serum and lipid. Choline is necessary for the biosynthesis of phosphatidylcholine, which is a main component of cell membranes. Phosphatidylcholine can be synthesized by the methylation of phosphatidyletha‐nolamine in liver cells. Phospholipid composition and incorporation of radiolabeled serine into phosphatidylserine or phosphatidylethanolamine were quite similar in R‐Y121B cho and its parental cells. However R‐Y121B cho cells had higher phosphatidylcholine synthesis activity from radiolabeled methionine than parental cells. These results indicate that choline requirement of mammalian cells depends on the activity of phosphatidylethanolamine methyltransferase.
The synergistic action of the copper chelator bathocuproine sulphonate and cysteine in enhancing growth of L1210 cells in vitro
Authors: Ishii Tetsuro , Bannai Shiro
Published: February 2005

The growth stimulating effect of a copper‐specific chelator, 2,9‐dimethyl‐4,7‐diphenyl‐1, 10‐phenonthroline‐sulfonic acid on mouse lymphoma L1210 cells in vitro has been studied. Since they are defective in cystine transport, these cells require cysteine for their growth in vitro. However, addition of cysteine does not greatly enhance cell growth because it is rapidly oxidized to cystine. We have observed that the copper chelator potently inhibited oxidation of cysteine in culture medium and that simultaneous addition of cysteine and the chelator greatly enhanced cell growth. The chelator alone stimulated cell growth slightly by stabilizing a small amount of cysteine effluxed from the cells to the medium. The chelator also enhanced the growth promoting activity of 2‐mercaptoethanol by stabilizing cysteine produced in the medium during culture. These results suggest that the chelator stimulates cell growth by inhibiting copper mediated oxidation of cysteine in culture medium.
Potassium‐induced reverse transformation of cells infected with a temperature‐sensitive transformation mutant virus
Authors: Lai Chiu‐Nan , Becker Frederick F.
Published: February 2005

High potassium concentrations altered the morphology and the ability to grow in soft agar in 6m2 cells, a clone of rat kidney cells infected with a temperature‐sensitive mutant of Moloney sarcoma virus. Approximately 60% of cells exhibited normal morphology in the presence of 94.8 mM potassium in isotonic medium at the temperature permissive for transformation, whereas 100% were normal at 72 mM potassium in hypertonic media. A significant reduction of growth in soft agar was also induced with these conditions. However, the synthesis ratio of virus‐specified transforming protein to marker viral protein was not altered. Na+K+ ‐ATPase might play a role in this reversetransformation process.
Functional properties of normal and inverted rat thyroid follicles in suspension culture
Authors: Tacchetti Carlo , Zurzolo Chiara , Monticelli Antonella , Nitsch Lucio
Published: February 2005

Rat thyroid follicles in both the normal and the inverted configuration have been cultured in suspension. It has been found that normal follicles (i.e., those having a structural organization similar to the follicles in vivo) express most functional properties of the thyroid gland. They trap iodide, synthesize, secrete, and iodinate thyroglobulin, and secrete thyroid hormone into the culture medium. On the contrary, in inverted follicles (i.e., those having a cell polarity reversed with respect to normal) we were unable to detect expression of some of these functions. We conclude that both the follicular organization and the proper cell polarity are needed for the full expression of thyroid functional properties.
Hematopoiesis in the rat: Quantitation of hematopoietic progenitors and the response to iron deficiency anemia
Authors: Kimura Hideo , Finch C. A. , Adamson John W.
Published: February 2005

To determine the quantitative effects of iron deficiency on erythropoiesis and to assess the response of erythroid progenitors to sustained anemia, we developed quantitative assays for various hematopoietic progenitors in the adult, Sprague‐Dawley rat including erythroid colony‐ and burst‐forming cells (CFU‐E and BFU‐E), granulocyte/macrophage colony‐forming cells (CFU‐GM), and megakaryocytic colony‐forming cells (CFU‐Meg). CFU‐E were cultured in methylcellulose and grew best in the presence of fetal calf serum. CFU‐GM, BFU‐E, and CFU‐Meg grew better in normal rat plasma and required the presence of pokeweed mitogen‐stimulated rat spleen cell conditioned medium. The numbers of progenitors and nucleated erythroblasts in total marrow were estimated by the ratios of radioactivity in the humerus to the total skeleton as determined by radioiron dilution. The numbers of progenitors and erythroblasts in the spleen were measured by simple dilution. Sustained anemia was brought about through chronic iron deficiency. The response to iron deficiency anemia (IDA) was monitored by the numbers of the various progenitors and their cell cycle characteristics as measured by the tritiated thymidine suicide technique. With IDA, the number of CFU‐E in the body (marrow plus spleen) was increased to 3.5 times control, whereas the numbers of BFU‐E and CFU‐GM were unchanged. There was no difference in the percentage of CFU‐E, BFU‐E, and CFU‐GM in DNA synthesis (68%, 19.4%, and 18.8%, respectively). With iron therapy of IDA, CFU‐E numbers in marrow began to decrease by day 1 and fell in a manner reciprocal to changes in the hematocrit. Marrow and spleen erythroblasts, 1.7 times control in IDA, increased further to 3.9 times control by the fourth day after iron administration. There was no change in BFU‐E or CFU‐GM numbers in response to iron repletion, although the fraction of progenitors increased in the spleen. Thus, IDA does not limit the increase in CFU‐E seen with anemia, but does restrict erythroid maturation. Furthermore, the increase in CFU‐E and the state of chronic anemia occur without detectable changes in the number or cell cycle state of the more primitive BFU‐E.
Anchorage‐independent growth of primary rat embryo cells is induced by platelet‐derived growth factor and inhibited by type‐beta transforming growth factor
Authors: Anzano Mario A. , Roberts Anita B. , Sporn Michael B.
Published: February 2005

Several growth factors implicated in the process of cellular transformation were tested for their ability to induce anchorage‐independent (AI) growth of primary rat embryo (RE) cells. Our results show that in the presence of 10% calf serum, platelet‐derived growth factor (PDGF), 1–30 ng/ml, has the strongest effect of all growth factors tested on Al growth. Type‐β transforming growth factor (TGF‐β), by itself, does not stimulate Al growth, and it inhibits the PDGF‐induced colony formation in a dose‐dependent manner (ED50 ∼ 0.03 ng/ml). Qualitatively similar responses are obtained by using an established line of fibroblasts, NIH 3T3 cells; the principal difference between the response of the primary cells and the established cell line is in colony‐forming efficiency in soft agar culture (15% and 90%, respectively, for growth of colonies > 1,500 μm2 diameter in the presence of 10 ng/ml PDGF). Since Al growth has been shown to correlate well with tumorigenicity in vivo, our results suggest that the transforming potential of PDGF in an appropriate responsive cell can be controlled not only through its interaction with its own receptor, but also by the presence of inhibitory factors such as TGF‐β.
Hexose transport in L6 rat myoblasts. II. The effects of sulfhydryl reagents
Authors: D'amore Tony , Lo Theodore C. Y.
Published: February 2005

The importance of sulfhydryl groups for hexose transport in undifferentiated L6 rat myoblasts was investigated. N‐ethylmaleimide (NEM) and p‐chloromer‐curibenzenesulfonic acid (pCMBS) inhibited 2‐deoxy‐D‐glucose (2‐DOG) transport in a time and concentration‐dependent manner. The inhibition produced by both reagents was virtually complete within 5 min, although neither reagent inhibited transport more than 70–80% regardless of the concentrations or incubation times used. Furthermore, the inhibition of 2‐DOG transport by pCMBS or NEM could not be prevented by simultaneous preincubation of cells with 20 mM D‐glucose or 20 mM 2‐DOG. This suggests that sulfhydryl groups required for transport are separate from the hexose binding and transport site. By comparing the effects of the membrane impermeant pCMBS to those of the membrane permeant NEM, cell surface sulfhydryl groups were shown to be essential for hexose binding and transport. In contrast to the inhibition of 2‐DOG transport, pCMBS and NEM had much less of an effect on 3‐O‐methyl‐D‐glucose (3‐OMG) transport. For example, 1 mM NEM inhibited 2‐DOG transport by 66%, whereas 3‐OMG transport was inhibited by only 7%. This supports the suggestion that these hexose analogues may be transported by different carriers. Kinetic analysis of transport shows that treatment of cells with 1 mM NEM or 1 pCMBS results in inactivation of the high affinity 2‐DOG transport system, whereas the low affinity transport system is unaffected. 3‐OMG is preferentially transported by the low affinity system.
Endocytosis of mannose‐6‐phosphate binding sites by mouse T‐lymphoma cells
Authors: Bourguignon Lilly Y. W. , Balazovich K. , Suchard Suzanne J. , Hindsgaul O. , Pierce M.
Published: February 2005

The endocytosis and intracellular transport of mannose‐6‐phosphate conjugated to bovine serum albumin (Man‐6‐P:BSA) by mouse T‐lymphoma cells were investigated in detail using several methods of analysis, both morphological and biochemical. Man‐6‐P:BSA was labeled with fluorescein or 125I and used to locate both surface and intracellular Man‐6‐P binding sites by light or electron microscopy, respectively. Incubation of cells with either fluorescent or 125I‐labeled Man‐6‐P:BSA at 0°C revealed a uniform distribution of the Man‐6‐P binding sites over the cell surface. Competition experiments indicate that the Man‐6‐P:BSA binding sites on the cell surface are the same receptors that can recognize lysosomal hydrolases. After as little as 1 min incubation at 37°C, endocytosis of Man‐6‐P binding sites was clearly observed to occur through regions of the plasma membrane and via vesicles that also bound anticlathrin antibody. After a 5–15‐min incubation of cells at 37°C, the internalized ligand was detected first in the cis region of the Golgi apparatus and then in the Golgi stacks using both autoradiography and immunocytochemistry to visualize the ligand. The appearance of Man‐6‐P:BSA in the Golgi region after 15–30 min was confirmed by subcellular fractionation, which demonstrated an accumulation of Man‐6‐P:BSA in light membrane fractions that corresponded with the Golgi fractions. After a 30‐min incubation at 37°C, the internalized Man‐6‐P binding sites were localized primarily in lysosomal structures whose membrane but not lumen co‐stained for acid phosphatase. These results demonstrate a temporal participation of clathrin‐containing coated vesicles during the initial endocytosis of Man‐6‐P binding sites and that one step in the Man‐6‐P:BSA transport pathway between plasma membrane and the lysosomal structure can involve a transit through the Golgi stacks.
Regulation of hexose transporters in chicken embryo fibroblasts: Stimulation by the phorbol ester TPA leads to increased numbers of functioning transporters
Authors: Yamada Kazuo , Tillotson Loyal G. , Isselbacher Kurt J.
Published: February 2005

As has been observed with many types of cultured cells, chicken embryo fibroblasts (CEF) when exposed to the tumor promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) develop a 3‐ to 4‐fold increase in hexose transport activity in 4 h. This increase in transport activity occurred despite a modest decline of 20% in [3H]leucine incorporation into acid insoluble fractions. Cycloheximide largely, but not completely, blocked the increase in transport activity during TPA exposure. The effects of TPA were somewhat similar to those of glucose starvation induced enhancement of hexose transport activity. Furthermore, with TPA there was no additive effect to that produced by glucose starvation. Plasma membrane enriched fractions were prepared from CEF treated with or without TPA. Membranes prepared from TPA exposed cells had a two‐fold enhancement of stereospecific D‐glucose transport activity as well as D‐glucose inhibitable [3H]cytochalasin B binding as compared to the membranes from control CEF. There was no effect on transport when membranes were exposed to TPA in vitro. These results provide strong evidence that TPA exposure leads to an increase in the number of functioning transporters, an effect largely requiring protein synthesis.
Phenotypic heterogeneity in lymphoblastic cell lines
Authors: Smith L. J. , McCulloch E. A.
Published: February 2005

Four T‐cell and two B‐cell lines from patients with lymphoblastic leukemia were examined with a panel of monoclonal antibodies for a variety of lineage and differentiation stage‐associated antigens during growth in liquid suspension. In five of the lines, markers normally associated with the granulopoietic lineage were found and the level of expression of these markers varied during culture. The sixth line, MOLT‐3, was examined in more detail. Using clonal selection it was found that phenotypic heterogeneity could readily be obtained and that subclones could be acquired that expressed a wide range of markers, typically in reproducible kinetic patterns, that were not detected on the parent clone. Previous results were confirmed showing that treatment with the drug 5‐azacytidine (5‐aza) prior to selection promoted the expression of the granulopoietic lineage associated antigen 80H.5 on MOLT‐3 subclones; however, treatment with 5‐aza appeared to inhibit substantially the expression of other novel markers by subclones following a second selection compared to untreated controls. It appears that the expression of markers normally associated with other lineages on leukemic lymphoblasts (lineage infidelity) may be characteristic of such lines and that phenotypically variant subclones expressing lineage infidelity can readily be obtained by clonal selection.
Necessity of transferrin for RNA synthesis in chick myotubes
Authors: Shoji Akiko , Ozawa Eijiro
Published: February 2005

Chick transferrin (Tf) is essential not only for growth and differentiation but also for the maintenance of chick myotubes in culture. Its removal from the culture medium gives rise to degeneration of the myotubes. The analysis of this process revealed that the removal resulted in decrease in total and messenger RNA content in the myotubes; this was mainly due to a decrease in RNA synthesis. Activity of in vitro RNA synthesis in isolated nuclei from myotubes cultured without Tf was lower than the activity in nuclei from myotubes cultured with Tf and increased with the addition of FeCl3. Although RNA degradation in myotubes was also enhanced following Tf removal, the degree was small. The synthesis of most proteins was reduced. In contrast to this, a few new proteins of unknown nature were synthesised in myotubes cultured in Tf‐free medium.

The role of Fe ion carried into the cells by Tf in promoting myogenic cell growth and differentiation and in preventing the myotubes from degeneration can be explained, at least in part, on the basis of its effect on RNA synthesis.

Since we have found that Fe is required for activation of RNA polymerase purified from embryonic muscles (Shoji and Ozawa, 1985b), these effects may be ascribed to this activating effect.
Endothelial cell injury in vitro is associated with increased secretion of an Mr 43,000 glycoprotein ligand
Authors: Sage Helene , Tupper Joan , Bramson Rachel
Published: February 2005

A novel, serum albumin‐binding glycoprotein of molecular weight (mw) 43,000 (43K protein) was initially purified from the culture medium of bovine aortic endothelial (BAE) cells (Sage, H., Johnson, C., and Bornstein, P., J. Biol. Chem. 259:3993–4007, 1984). Its secretion by normal mesenchymal cells and by transformed cells of both ectodermal and endodermal origin suggested a general role in cellular function. To examine the effect of sublethal injury in vitro on the biosynthesis of 43K protein, BAE cells were exposed to endotoxin. At concentrations which produced minimal cell detachment and lysis, the cells secreted 70–100% more protein compared to control cultures, and the relative increase in 43K protein over total protein was approximately three‐fold. A second type of cellular injury, manifested by rapid cellular proliferation and migration in response to sparse plating density (a condition that we have termed ‘culture shock’), was also accompanied by a significant increase in the secretion of 43K protein.

Pulse‐chase studies revealed that the initial product secreted within 1.5 h was of Mr 38,000, and that between 6 and 21 h this molecule was converted to the final form of Mr 43,000. The 43K protein was not associated with RNA or glycosaminoglycan, but appeared to be linked to complex oligosaccharides containing peripheral sialosyl residues. Treatment with tunicamycin produced lower mw forms that displayed reduced affinity for albumin. By immunologic criteria, peptide mapping, and amino acid analysis, the 43K protein was shown to be structurally distinct from several proteins of Mr 40,000–50,000 associated with endothelium or with serum, including tissue factor, a plasminogen anti‐activator, and several apolipoproteins. In addition, the 43K protein was not present in the extracellular matrices of endothelial, fibroblastic, or smooth muscle cells, nor was it found in plasma, serum, platelet releasate, or alveolar lavage fluids.

These studies identify a unique Mr 43,000 glycoprotein that is associated with cellular stress or injury in vitro. As a secreted but nonmatrix macromolecule, this protein may be part of a ‘survival kit’ used by the endothelium to cope with cellular injury.
Internalization of transforming growth factor‐β and its receptor in BALB/c 3T3 fibroblasts
Authors: Massagué Joan , Kelly Brenda
Published: February 2005

The fate of 125I‐labeled transforming growth factor‐β (125I‐TGFβ) after binding to its cells surface receptor has been investigated in BALB/c 3T3 mouse fibroblasts. Binding of 125I‐TGFβ to cellular receptors at 4°C is pH‐sensitive, being markedly decreased at pH < 6. Most (∼ 90%) of the 125I‐TGFβ bound to cells at 4°C can be removed by a brief treatment with acidic medium but is converted into an acid‐resistant state rapidly after shifting the cells to 37°C. Cell‐bound 125I‐TGFβ is degraded at 37°C and the degradation products are released into the medium. The lysosomotropic bases chloroquine, methylamine, and ammonium and the carboxylic ionophore monensin inhibit the degradation and release of 125I‐TGFβ from the cells. Cells allowed to accumulate 125I‐TGFβ intracellularly by the action of chloroquine or monensin were treated with the bifunctional agent disuccinimidyl suberate in the presence of detergent Triton X‐100; this treatment caused the cross‐linking of internalized 125I‐TGFβ with the 280‐kilodalton TGFβ receptor component. Under conditions in which sustained binding and degradation of saturating 125I‐TGFβ concentrations occurs, there is no marked decrease in the binding capacity of the cells even when protein synthesis is blocked with cycloheximide. These results indicate that after TGFβ binding the TGFβ:receptor complex becomes rapidly internalized and that TGFβ is directed towards lysosomes where it is degraded and released. However, the cell surface is replenished with TGFβ receptors recycled after internalization or supplied by a large intracellular pool.
Stress induces an increased hexose uptake in cultured cells
Authors: Warren A. P. , James M. H. , Menzies D. E. , Widnell C. C. , Whitaker‐Dowling P. A. , Pasternak C. A.
Published: February 2005

Temperature‐sensitive mutants have revealed a region of the herpes simplex virus 1 genome that affects both the uptake of hexose and the synthesis of heat shock proteins. Other inducers of heat‐shock proteins, namely heat shock itself and arsenite, likewise induce an increased uptake of hexose. The increased uptake, like that induced by insulin, is insensitive to the presence of actinomycin D or cycloheximide. It is concluded that an increased hexose uptake, reflecting an activation or relocation of existing hexose transport protein, is general biochemical response of stressed cells.
Ultrastructural studies of the intracellular translocation of endocytosed alpha‐foetoprotein (AFP) by cytochemistry and of the uptake of 3H‐arachidonic acid bound to AFP by autoradiography in rat rhabdomyosarcoma cells
Authors: Geuskens Maurice , Naval Javier , Uriel José
Published: February 2005

A covalent conjugate of alpha‐foetoprotein (AFP) and horseradish peroxidase (HRP) has been used to follow, at the ultrastructural level, the pathway of AFP uptake and translocation in a rat rhabdomyosarcoma cell line. The cells were incubated for several times at 4°C and/or 37°C, and fixed. AFP‐HRP was found to enter the cells via coated pits and receptosomes and to move to tubular elements of the trans‐reticular portion of the Golgi. Some observations suggest that AFP can be recycled back to the cell surface. On the other hand, the cells were incubated with a noncovalent conjugate of AFP and 3H‐arachidonic acid [3H‐(20:4)], and the uptake of the fatty acid molecules studied by ultrastructural autoradiography. The cytoplasmic labeling, very low after an incubation in the presence of [3H‐(20:4)]‐AFP for 2 hours at 4°C, increased rapidly after transfer of the cells for 5 minutes to 37°C. These observations support the hypothesis that AFP plays a role in the intracellular delivery of polyunsaturated fatty acids.
Functional differences in the interactions of glycosylation‐deficient cell lines with fibronectin, laminin, and type IV collagen
Authors: Hughes R. Colin , Mills Gary
Published: February 2005

Fibronectin isolated from the conditioned medium of monolayer cultures of baby hamster kindey (BHK) cells and several ricin‐resistant (Ric®) mutants derived from them express differences in N‐glycosylation. The asparaginelinked oligosaccharides of BHK cell‐derived fibronectin consist largely of complex chains, whereas hybrid and/or high‐mannose chains are present in the fibronectins of mutant cell lines. The fibronectins exhibiting different glycosylation patterns are incorporated to similar extents into the cell‐layer of human skin fibroblasts. In contrast, mutant cells retain significantly less endogenously produced fibronectin than BHK cells and also incorporate less human cellular fibronectin into a pericellular matrix. In vitro adhesion assays show that mutant cells attach to and spread relatively poorly on fibronectinor type IV collagen‐coated substrata but interact as well as do BHK cells with a laminin substratum. These results indicate that asparagine‐linked oligosaccharides of fibronectin are not required for the binding and incorporation of the molecule into cell layers, but, as constituents of other cellular glycoproteins, they do modulate the ability of BHK cells to interact with some matrix components.
Heparin protects basic and acidic FGF from inactivation
Authors: Gospodarowicz D. , Cheng J.
Published: February 2005

The ability of heparin or that of hexuronyl hexosaminoglygan sulfate (HHS‐4) to protect basic or acidic fibroblast growth factor (FGF) from acid or heat inactivation has been analyzed. Both freshly prepared basic and acidic FGF stimulate the growth of baby hamster kidney (BHK‐21) cells exposed to medium supplemented with transferrin and insulin. Freshly prepared basic FGF was 10 fold more potent than acidic FGF. The addition of heparin to the medium decreased the potency of basic FGF while it potentiated that of acidic FGF. Upon storage of FGF at −80°C, a decline in potency of both basic and acidic FGF was observed. Heparin, when added to the medium, potentiated their activities, which became similar to that of freshly prepared basic FGF. In order to test whether heparin could protect basic or acidic FGF from inactivation, both mitogens were exposed to acid conditions (1% trifluoroacetic acid, pH 1.08, 2 h) or heat (65°C, 5 min) which inactivate basic or acidic FGF. When exposed to such treatment in the presence of heparin or HHS‐4, basic and acidic FGF retained their potency. The effect of heparin and HHS‐4 on the bioactivity of basic and acidic FGF is truly of a protective nature, since they had no effect when added after inactivation of the mitogens. Potentiation of the bioactivity of the protected mitogens or of the inactivated one could only be observed when cells were exposed to high heparin or HHS‐4 concentrations. This indicates that heparin and HHS‐4, in addition to protecting FGF from inactivation, also acts at another locus, as yet unidentified.
Thrombin‐induced chemotaxis and aggregation of neutrophils
Authors: Bizios Rena , Lai Linda , Fenton John W. , Malik Asrar B.
Published: February 2005

Thrombin‐induced neutrophil chemotaxis and aggregation were studied using cells isolated from either human or sheep blood. Sheep neutrophils (108 cells/ml) exhibited maximum chemotactic migration towards 10−8 M human α ‐thrombin, 10−8 M γ‐thrombin (which lacks the fibrinogen site), and 10−12 MD‐Phe‐Pro‐Arg‐CH2‐α‐thrombin (catalytically inactive thrombin). Chemotactic responses of the same magnitude were obtained with human neutrophils (108 cells/ml). The chemotactic responses to thrombin were comparable to those obtained with diluted (1:200 v/v) zymosan activated serum (ZAS) and 10−11 M FMLP. Premixing of the thrombin forms with hirudin in 1:1 stoichiometric amounts abolished the chemotaxis but not chemokinesis Aggregatory responses of human and sheep neutrophils were comparable for ZAS, α‐thrombin, and γ‐thrombin. The responses of both human and sheep neutrophils to D‐Phe‐Pro‐Arg‐CH2‐α‐thrombin were attenuated, indicating that the proteolytic site may be involved in the aggregatory response. The results suggest that thrombin‐induced neutrophil chemotaxis and aggregation are mediated by different mechanisms, since chemotaxis is a catalytically independent response whereas aggregation is an active site independent response.
Glucocorticoid agonists as well as antagonists are effective inducers of mouse mammary tumor virus RNA in mouse mammary tumor cells treated with inhibitors of ADP‐ribosylation
Authors: Johnson George S. , Ralhan Ranju
Published: February 2005

Glucocorticoids increase expression of specific genes by a mechanism involving binding to and “activation” of a specific receptor protein. Other steroids, such as RU 486, bind to the glucocorticoid receptor but the resultant steroidreceptor complex is unable to activate glucocorticoid sensitive genes. In the present study we have observed that steroid regulation of the glucocorticoidregulated mouse mammary tumor virus (MMTV) genome in cultured mouse mammary tumor cells is altered by treatment of the cells with inhibitors of (ADP‐ribose)n synthetase. The ability of glucocorticoid agonists to increase MMTV is about 2‐fold increased by the inhibitor treatment. Interestingly, RU 486 and other steroids that are normally inactive in control cells are very good inducers of MMTV in the treated cells. This alteration in MMTV expression is associated with a 37% increase in nuclear binding of the glucocorticoid, triamcinolone acetonide, and also RU 486 in the inhibitor‐treated cells. Steroids that do not bind to the glucocorticoid receptor are not inducers in control or in treated cells. The results point to a role for ADP‐ribosylation of proteins as a negative regulator of MMTV expression and suggest a mechanism for activation of steroid‐sensitive genomes.