bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding and internalization of heparin by vascular smooth muscle cells
Authors: Castellot John J. , Wong Kent , Herman Brian , Hoover Richard L. , Albertini David F. , Wright Thomas C. , Caleb Benjamin L. , Karnovsky Morris J.
Published: February 2005

Previous work from our laboratory has demonstrated that heparin specifically inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. In this paper, we examine the binding and mode of internalization of heparin by smooth muscle cells. For these studies, radiolabeled and fluoresceinated (FITC) heparin probes were synthesized that retained their antiproliferative capacity. Binding of 3H‐heparin to these cells occurs via specific, high‐affinity binding sites (Kd = 10−9 M, 100,000 binding sites per cell). Approximately 80% of the heparin bound to the cell surface was shed into the culture medium within 2 hr. The heparin that was left on the cell surface was internalized with biphasic kinetics. Approximately 50% of the bound material was internalized within 2 hr. After this initial rapid uptake, the rate slowed substantially, with the remaining heparin requiring 1–2 days to be internalized. Binding and uptake of FITC heparin was monitored using video image intensification fluorescence microscopy. When smooth muscle cells were exposed to FITC heparin at 4°C, a diffuse surface staining pattern was observed. After warming the cells to 37°C, intensely fluorescent vesicles were seen superimposed over the diffuse surface staining within 2 min. After 15 min at 37°C, numerous large punctate vesicles were seen inside the cell. After 2 hr these vesicles had concentrated in the perinuclear region. This pattern of uptake, when considered along with the presence of specific, high‐affinity binding sites and the initial rapid uptake of 3H‐heparin, suggests that heparin enters smooth muscle cells by both receptor‐mediated and other endocytic pathways.
Effect of heparin on vascular smooth muscle cells. I. Cell metabolism
Authors: Castellot John J. , Cochran David L. , Karnovsky Morris J.
Published: February 2005

Previous work from our laboratory has shown that heparin inhibits the proliferation of vascular smooth muscle cells in vivo and in vitro. The mechanism of action of this glycosaminoglycan is unknown. In this communication, we have examined the antiproliferative effect of heparin on smooth muscle and other cell types, and have investigated several aspects of heparin on smooth muscle cell metabolism. Smooth muscle and closely related cell types from several species, including human, were much more sensitive to heparin than any other cell type tested, including primary and established cell lines, normal and transformed cell pairs, fibroblasts epithelial, and endothelial cells. Flow microfluorimetric analysis of cell cycle distribution indicated that heparin blocked either the Go → S transition or a very early S‐phase event in smooth muscle cells. Heparin rapidly inhibited DNA and RNA synthesis, but did not affect the rate of protein synthesis. The decrease in nucleic acid synthesis could be accounted for by an inhibition of thymidine and uridine uptake. Interestingly, heparin did not block amino acid or glucose transport. Although no change in the overall rate of protein synthesis was observed in the presence of heparin, we noted at least two changes in the synthesis of specific proteins by smooth muscle cells: two 35,000‐dalton proteins which appeared in the culture medium of heparin‐treated cells, and the transient disappearance of a 48,000‐dalton protein in the substrate attached material of smooth muscle cells exposed to heparin. The role of the observed changes in smooth muscle cell metabolism is yet to be determined, but they may provide valuable clues to the molecular mechanisms controlling the antiproliferative activity of heparin.
Effect of potassium depletion of cells on their sensitivity to diphtheria toxin and pseudomonas toxin
Authors: Sandvig Kirsten , Sundan Anders , Olsens Sjur
Published: February 2005

When Vero cells were depleted of potassium, the cells were protected against diphtheria toxin, Potassium deletion of Vero cells strongly reduced the binding of the toxin to cell surface receptors. Likewise, potassium depleted L‐cells were protected against pseudomonas toxin. Diphtheria toxin binding was completely restored upon addition of potassium to the cells. This restoration was not prevented by inhibition of protein synthesis by cycloheximide. When cells were depleted of potassium in the presence of metabolic inhibitors, and then treated with diphtheria toxin, protein synthesis was reduced to the same extent as in cells with normal intracellular level of potassium. The results indicate that potassium depletion of Vero cells reduces the ability of the cells of bind diphtheria toxin by an ATP requiring process, and that binding, endocytosis and transfer of diphtheria fragment A across the membrane may occur at low intracellular levels of potassium.
Insulin binding and vesicular ingestion in capillary endothelium
Authors: Solenski Nina J. , Williams Stuart K.
Published: February 2005

Capillary endothelium can actively regulate vascular permeability of various serum proteins. Hormones such as insulin must interact with this capillary barrier in order to reach their respective target tissues. We have studied the binding and subsequent internalization of 125I‐insulin in both native (freshly isolated) and primary cultured capillary endothelium derived from rat epididymal fat pads. Insulin association with the endothelium, internalization and degradation differed between freshly isolated and primary cultured capillaries. Specific binding in freshly isolated and cultured capillaries was temperature dependent, and was competitively inhibited in the presence of unlabelled insulin. Primary cultures of capillaries grown to confluence did not exhibit specific binding of insulin. Despite the lack of specific receptors for insulin, cultured cells vesicularly internalized insulin. Greater than 50% of the total associated insulin was not degraded by cultured endothelium. Morphological examinations using ferritin labelled insulin localized insulin associated to the capillary endothelial cell membrane and sequestered within pinocytotic vesicles. Incubation of freshly isolated capillaries with insulin stimulated the fluid phase endocytosis of 14C‐sucrose; however, insulin had no effect on fluid phase endocytosis in cultured capillaries. These results indicate that capillary endothelium, isolated from rat epididymal fat, exhibit specific receptors for insulin. Binding of insulin to the capillary membrane is followed by internalization into cytoplasmic vesicles and partial degradation.
Fibroblast products adsorb to the culture substrate and stimulate growth of human umbilical vein endothelial cells
Authors: Kan Mikio , Kato Miwa , Yamane Isao
Published: February 2005

Human umbilical vein endothelial cells grew with a doubling time of approximately 20 hr in medium conditioned by human diploid fibroblasts and supplemented with 10% fetal bovine serum, whereas the cells did not grow substantially in the non‐conditioned serum supplemented medium. Production of the fibroblast‐derived activity required the presence of insulin, EGF, or PDGF. The fibroblast derived‐factor adsorbed to native culture dishes or dishes coated with gelatin and collagen. The adsorbed activity was resistant to treatment with 1% Triton X‐100, and was abolished by treatment with serine proteases. Further, the extracellular matrix produced by the fibroblasts also showed growth‐stimulating activity. Fibroblast‐derived factors may play a role in vascularization processes during wound healing, inflammation and normal development.
Release of galactosyltransferase from peritoneal macrophages during acute inflammation
Authors: Hopper K. , Hollister W. , Davey R. , Semler A.
Published: February 2005

Peritoneal cells harvested from mice injected with Salmonella enteritidis or thioglycollate released large amounts of galactosyltransferase (GT), but not sialyltransferase, into their culture supernatants. Maximum release of GT (using ovalbumin as acceptor) occurred from cells harvested 2–4 days after primary injection, but little GT was released from cells elicited by a secondary injection of salmonella or ovalbumin in sensitised mice or during intraperitoneal allogeneic reactions. Enzyme release in culture did not parallel GT levels in serum. Most enzyme was released by large, poorly adherent, macrophageenriched, Fc receptor‐bearing peritoneal cells of low density. Normal monocytes, bone marrow cells, and platelets also produced large amounts, and normal spleen cells or polymorphonuclear leukocytes moderate amounts, of GT. Lymphocytes, dead cells, mast cells, red blood cells, or whole populations of lymph node and thymus cells released very low levels of enzyme. Very little GT was bound to the cell surface and was not passively absorbed from serum or platelets. Release of GT was prevented at 4°C but was not markedly affected by a variety of metabolic inhibitors except pretreatment of the cells with thrombin, which increased release and trypsin which decreased release.
A role for soluble cAMP phosphodiesterases in differentiation of 3T3‐L1 adipocytes
Authors: Elks M. L. , Manganiello V. C.
Published: February 2005

Differentiatio of 3T3‐L1 adipocytes, monitored by accumulation of neutral lipid and by increase in α‐giycerophosphate dehydrogenase activity, is accelerated by incubation of confluent 3T3‐L1 fibroblasts in media containing insulin, dexamethasone and isobutylmethylxantine (IBMX). IBMX inhibits cyclic nucleotide phosphodiesterases as well as the binding of adenosine to its recetor. Agents with relatively specific effects were utilized to examine the role of IBMX in differentiation. Ro 20–1724, a selective inhibitor of soluble cAMP phosphodiesterase activities, was as effective as IBMX in increasing α‐glycerophosphate dehydrogenase activity and fat deposition. Neither cilostamide, which inhibits particulate but not soluble cAMP phosphodiesterase activities, 8‐phenyltheophylline, an adenosine receptor antagonist with little inhibitory effect on phosphodiesterase activities, nor N6‐(R phenyl‐isopropyl) adenosine (PIA), a potent adenosine receptor agonist, were effective in promoting differentiation. In addition, we find that maximal increases in α‐glycerophosphate dehydrogenase activity and lipid accumulation were observed when differentiation was initiated in the presence of 10 nM dexamethasone. These data suggest that inhibition of soluble cAMP phosphodiesterase activity and subsequent alterations in cAMP may play an important role in the mechanism whereby IBMX enhances differentiation of 3T3‐L1 cells.
Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells
Authors: Willey James C. , Laveck Moira A. , McClendon Irene A. , Lechner John F.
Published: February 2005

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum‐free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L‐epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPi is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, α‐ (difluoromethyl)‐ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the β‐adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF‐stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.
Selective expression of brush border hydrolases by mouse Peyer's patch and jejunal villus enterocytes
Authors: Smith Michael W.
Published: February 2005

Developmental profiles describing the expression of lactase, α‐glucosidase, and alkaline phosphatase activities have been determined quantitatively in mouse jejunal enterocytes during migration over villi and Peyer's patch lymphoid tissue. The predicted maximal lactase and α‐glucosidase activities expressed by enterocytes migrating over Peyer's patch follicles were about one‐quarter and one‐half of values found in control villi. Alkaline phosphatase activity was, on the other hand, one third greater in Peyer's patch compared with villus enterocytes. Expression of lactase and α‐glucosidase activities was initially less in enterocytes migrating along interfollicular compared with control villi. Subsequent increase in hydrolase activities occurred during the later stages of enterocyte migration over interfollicular villi. Lactase activity in athymic mice Peyer's patch enterocytes was identical to that recorded for control mice. The corresponding value for villus lactase was, however, only half that found in control tissue. Factors produced locally in lymphoid follicles are probably responsible for selective effects on enterocyte differentiation.
Effects of pulsed electromagnetic field on growth and differentiation of embryonal carcinoma cells
Authors: Akamine Takuya , Muramatsu Hisako , Hamada Hiroshi , Sakou Takashi
Published: February 2005

A murine embryonal carcinoma cell line (F9) was used to examine the effect of a pulsed electromagnetic field on the growth and differentiation of malignant cells. The cells can be induced to differentiate into parietal endodermal cells by treatment with retinoic acid. The pulsed electromagnetic field (1 Gauss and 10 Gauss) promoted the growth of embryonal carcinoma cells in both the presence and absence of retinoic acid. The pulsed electromagnetic field was also found to inhibit retionic acid‐induced differentiation, when the degree of differentiation was based on morphological criteria or on the production of plasminogen activator.
A gene‐controlling response of bone marrow progenitor cells to granulocyte‐macrophage colony stimulating factors
Authors: Lazar Gary S. , Quon Diana H. , Lusis Aldons J.
Published: February 2005

The production of granulocytes and macrophages from progenitor cells in the bone marrow is controlled, in part, by a family of humoral regulators, termed colony stimulating factors (CSF). We have examined genetic factors controlling this process using in vitro cloning techniques. The inbred mouse strain LP/J showed elevated colony formation (CFU‐C) in response to one subtype of CSF (G, M‐CSF) compared to other strains of mice examined including the strain C57BL/6J. This variation resulted in a shift to the left of the CFU‐C dose‐response curve for LP/J. No difference between LP/J and C57BL/6J was seen with another subtype of CSF (CSF‐1). Maximal CFU‐C response was similar in the two mouse strains with both types of CSF, and mixing experiments with both types of CSF gave the same maximal level of colony formation as the individual CSF. (C57BL/6J x LP/J)FI progeny exhibited a CFU‐C dose‐response curve to CSF‐2 that was intermediate between the parental types, indicating additive inheritance. Genetic analysis of backcross progeny suggested that the variation in CFU‐C response is probably determined by a single primary gene, although the variability of the colony formation assay has complicated interpretation of genetic studies. These results suggest that CSF‐1 and G, M‐CSF act independently on a single bone marrow progenitor cell population. The properties of the genetic variation for G, M‐CSF response are consistent with an alteration in cellular receptors for G, M‐CSF.
Inhibition of epidermal growth factor‐induced phosphorylation by trifluoperazine
Authors: Ross Alonzo H. , Damsky Caroline , Phillips Paul D. , Hwang Frances , Vance Patricia
Published: February 2005

We studied the effects of a series of drugs on A431, a cell line with well‐characterized growth factor requirements and epidermal growth factor (EGF) receptors. The major [32PO4]‐labeled protein immunoprecipitated with antiphosphotyrosine antibodies from EGF‐stimulated A431 cells was the EGF receptor. Both the quantity of [32PO4]‐labeled proteins were reduced by the addition during EGF stimulation of trifluoperazine (TFP). TFP had little effect on the binding, internalization, and processing of [125I]‐EGF. In addition to the effects on phosphorylation, TFP inhibited cell growth both in the presence and absence of EGF. Morphologically, TFP blocked EGF‐induced ruffling. TFP did not alter the EGF‐ stimulated phosphatidylinositol turnover. In an in vitro experiment using A431 cell membranes, TFP did not inhibit phosphorylation of the EGF receptor.
Ammonia production by IMR‐90 fibroblast cultures: Effects of ammonia on glutathione, γ‐glutamyl transpeptidase, lysosomal enzymes, and cell division
Authors: Takahashi Shizuko , Nakagawa Sachiko , Zeydel Maria , Bhargava Girija
Published: February 2005

γ‐Glutamyl transpeptidase has multi‐catalytic activities. It degrades glutathi‐one and can produce ammmonia from glutamine. The present study was designed to examine whether the decreased cell proliferation, cellular gluta‐thione content and concurrent increase in ammonia production in senescent cells in culture are the result of increased γ‐glutamyl transpeptidase activity. We used IMR‐90 fibroblast and 3T3 LI preadipocyte cultures. The cellular glutathione content depended upon cell proliferation and cell density. The glutathione content was higher in cells at logarithmic growth, and lower at stationary growth or post confluency; dead cells had no detectable glutathione by the method currently used. The glutathione content was minimal in “old” IMR‐90 cells, regardless of cell density. On the other hand, an increase occurred in the unit number of molecules of bound 5‐iodoacetoamidofluorescein, an active‐site directed stoichiometric inhibitor of transpeptidase. That result corresponded favorably with the increased enzyme activity, suggesting that the number of enzyme molecules per cell was increased. The inhibition of ammonia production of the cultures by inhibition of γ‐glutamyl transpeptidase by 5‐iodoacetoamidofluorescein and reversible inhibition of ammonia production by a serine‐borate mixture were consistent with our postulate. Addition of NH4Cl (0.1 mM) to IMR‐90 cultures caused increased activities of transpeptidase and some of the lysosomal enzymes; concurrently, the amount of cellular glutathione and the number of cell divisions decreased. This suggests that the increased ammonia production presumably resulting from glutaminase activity of the observed increase of transpeptidase may profoundly affect certain cellular functions.
Sulfate transport in human lung fibroblasts (IMR‐90)
Authors: Elgavish Ada , Smith Jeffrey B. , Pillion Dennis J. , Meezan Elias
Published: February 2005

Sulfate transport in a fibroblast cell line derived from human lung (IMR‐90) occurred mainly via high‐ and low‐affinity, SITS‐sensitive pathways and to a lesser extent by an SITS‐insensitive mechanism. In low‐ionic‐strength media (sucrose substituted for salts) the apparent Km of the carrier‐mediated sulfate influx was 1 mM. At 0.3 mM, the sulfate concentration normally found in human serum, the contribution of the SITS‐insensitive pathway was negligible. In physiological salts solution, an SITS‐sensitive, high‐affinity (Km 34 ± 14 μM) sulfate influx system was observed at extracellular sulfate concentrations less than 100 μM. Between 100 and 500 μM sulfate, the range normally found in human serum, sulfate influx occurred via an SITS‐insitive, lowaffinity pathway and to a small extent by an SITS‐insensitive mechanism. Extracellular chloride inhibited the influx and stimulated the efflux of sulfate. Bicarbonate and thiosulfate inhibited sulfate influx but had no effect on sulfate efflux. Phosphate, arsenate, or Na+ did not affect sulfate uptake. These results indicate that in human lung fibroblast IMR‐90 cells sulfate is transported mainly via an SO42−/Cl‐ exchange system independent of the phosphate or Na+ transport. Since sulfate concentration as high as 50 mM only slightly increased sulfate efflux, SO42−/SO42− exchange is probably a minor component of sulfate uptake.
Expression of glucagon sensitivity by transformed MDCK cells normally unresponsive to glucagon: Early commitment to differentiation
Authors: Beckner Suzanne K. , Lin Michael C.
Published: February 2005

A cloned line of canine kidney cells (MDCK) transformed with Harvey murine sarcoma virus, in contrast to the parental, untransformed line, expressed glucagon sensitivity only under controlled culture conditions. The glucagon sensitivity of transformed MDCK cells appeared after 10 days of culture if plated at less than 100,000 cells/dish or after 3 days if cells were plated at greater than 300,000 cells/dish. As there was no effect of conditioned medium from glucagon‐sensitive cells on insensitive cells, media components seemed not to be involved in this phenomenon. Glucagon sensitivity appeared more readily in defined as opposed to serum‐containing medium. In fact, as little as 2% fetal bovine serum inhibited the expression of glucagon sensitivity when included in defined medium over the course of the experiment. Furthermore, when transformed MDCK cells were exposed to serum for only the first 24 hr of culture, glucagon sensitivity on day 11 was identical to that of cells exposed to serum throughout the entire experiment. In contrast, exposure to serum later in culture (days 4–8) had no inhibitory effect on the expression of glucagon sensitivity on day 11. The data suggest that differntiation, or glucagon sensitivity, occurs when transformed, glucagon‐insensitive cells achieve a critical high density and that differentiation is sensitive to inhibition by serum only during the first 24 hr of culture.
Phosphorylation of rabbit skeletal muscle myosin in situ
Authors: Moore Russell L. , Houston Michael E. , Stull James T. , Iwamoto Gary A.
Published: February 2005

Myosin light chain (P light chain) is phosphorylated by Ca2+. calmodulindependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow–twitch soleus and fast‐twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast‐twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin‐activated ATPase activity, may be related to isometric twitch potentiation.
Subcellular localization of histone messenger RNAs on cytoskeleton‐associated free polysomes in HeLa S3 cells
Authors: Zambetti Gerard , Stein Janet , Stein Gary , Schmidt Warren
Published: February 2005

We have examined the subcellular distribution of histone mRNA‐containing polysomes in HeLa S3 cells to assess the possible relationship between localization of histone mRNAs and the regulation of cellular histone mRNA levels. The distribution of histone mRNAs on free and membrane bound polysomes was examined as well as the association of histone mRNA‐containing polysomes with the cytoskeleton. The subcellular localization of histone mRNAs was compared with that of HLA‐B7 mRNAs which encode a cell surface antigen. Histone mRNAs were localized predominantly on the free polysomes, whereas the HLA‐B7 mRNA was found almost exclusively on membrane bound polysomes. However, both species of mRNA were found associated with the cytoskeleton. Interruption of DNA synthesis by hydroxyurea treatment resulted in a rapid and selective destabilization of histone mRNAs in each subcellular fraction; in contrast, the stability of HLA‐B7 mRNA appeared unaffected. The results presented confirm that histone mRNAs are predominantly located on non‐membrane bound polysomes and suggest that these polysomes are associated with the cytoskeletal framework.
Rat fibroblasts expressing high levels of human c‐myc transcripts are anchorage‐independent and tumorigenic
Authors: Pellegrini Sandra , Basilico Claudio
Published: February 2005

The c‐myc oncogene has been implicated in the genesis of tumors as it has been found to undergo rearrangements and structural alterations in several types of neoplasms, However, the molecular mechanism of its involvement in transformation is still unclear as is the function of its protein product. We have studied the biological activity of the normal human c‐myc oncogene in a well‐characterized rat fibroblast line, F2408. The human gene, deleted of its 5′ non‐coding exon, was placed under the control of the Polyoma virus DNA regulatory region, linked to a dominant selectable marker (neomycin‐resistance gene) and transfected into F2408 cells. Several neoR colonies were assayed for ability to grow in agar suspension, and the steady‐state levels of mRNA transcripts of the exogenous c‐myc gene were analyzed by Northern blot. While the greatest majority of these cell populations are phenotypically normal and express low levels of human myc transcripts, the rare neoR clones which display anchorage‐independence express the human gene at high levels. This correlation was confirmed by the analysis of two neoR lines which contain low levels of c‐myc RNA but gave rise to agar colonies at a frequency of about 10−5. These agar derivatives were found to have greatly increased levels of c‐myc mRNA when compared to the respective parental lines. The clones expressing high levels of myc RNA were found to be tumorigenic upon inoculation into young syngeneic rats. These results suggest that constitutive high expression of the normal c‐myc gene is sufficient to confer anchorage‐independence and tumorigenic potential to established rat fibroblasts.
The effects of epidermal growth factor and the state of confluence on enzymatic activities of cultured rat liver epithelial cells
Authors: Tsao Ming‐Sound , Earp H. Shelton , Grisham Joe W.
Published: February 2005

In cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH‐diaphorase (NADH‐D), pyruvate kinase (PK), glucose‐6 phosphate dehydrogenase (G6PD), gamma‐glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH‐D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH‐D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF‐directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF‐mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two‐to threefold higher capacity to bind [125I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth‐state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF‐like factors may contribute to the high rate of glycolysis in certain neoplasms.
Reduced pinosome formation in a cell‐fusion‐impaired mutant of mouse lymphocytic cell line L5178Y
Authors: Pauw Peter G. , Biagi Karen G. , Stadler Joan K.
Published: February 2005

Accumulation and release of a fluid‐phase marker ([14C]‐sucrose) were studied in a subline of the mouse lymphocytic cell line L5178Y and in a polyethylene glycol‐resistant, intercellular‐fusion‐impaired mutant of this line. The mutant was found to accumulate [14C]‐sucrose at a significantly slower rate than the parent. Analysis of release of preloaded label shows that the reduced rate of accumulation is due to a correspondingly low level of internalization, i.e., pinosome formation, rather than to a reduction in delivery of label to lysosomes. Cell‐to‐cell fusion and pinosome formation both involve a fusion event initiated at the extracellular surface of the plasma membrane, and we propose that the coordinate reduction in both processes suggests that they are mechanistically related.
Heat shock proteins within the mammalian cell cycle: Relationship to thermal sensitivity, thermal tolerance, and cell cycle progression
Authors: Rice Glenn , Laszlo Andrei , Li Gloria , Gray Joe , Dewey William
Published: February 2005

We have measured endogenous and induced rates of 70‐kD, 89‐kD, and 110‐kD heat shock proteins in highly pure G1‐, S‐, or G2‐M phase fractions of Chinese hamster fibroblasts (CHO) separated by fluorescence‐activated cell sorting (FACS). Relative rates of synthesis of all three polypeptides as measured by two‐dimensional gel electrophoresis were similar throughout the cell cycle, and therefore, endogenous levels were unlikely to explain the thermal sensitivity of S‐phase cells. Distinct heterogeneity in induced rates of these polypeptides was noted in all phase fractions. Enhanced rates of 70‐kD polypeptide were measured in S and G2‐M as compared to G1 following heat shock. Little increase in either the 89‐kD or 110k‐kD heat shock proteins was observed in heated G1 cells. This heterogeneity in induced rates of synthesis was in contrast to the similarity in thermal tolerance expression kinetics between each phase. Finally, enhanced synthesis of these polypeptides appeared unrelated to regulation of either heat‐induced cell cycle delay or to the resumption of phase‐specific progression after heat shock as measured by simultaneous flow cytometric measurement of incorporated BrdUrd and DNA content.
Binding and metabolism of leukotriene B4 by neutrophils and their subcellular organelles
Authors: O'Flaherty Joseph , Kosfeld Scott , Nishihira Jun
Published: February 2005

The subcellular distribution of leukotriene (LT)B4 binding and metabolizing sites was investigated in human neutrophils. Cells were disrupted by nitrogen cavitation and fractionated by Percoll density gradient centrifugation to yield cytoplasm, membranes, azurophilic granules, and specific granules. Only membrane fractions contained high affinity [3H]LTB4 binding sites. Binding of radiolabeled ligand to membranes was rapid, reversible, and saturable; it was blocked by a series of LTB4 analogues at concentrations corresponding to their respective potencies in (1) blocking [3H]LTB4 binding to whole cells and (2) stimulating neutrophil degranulation responses. In contrast, [3H]LTB4 was metabolized by fractions enriched with markers for cytoplasm plus endoplasmic reticulum. The metabolic activity was sedimented by ultracentrifugation, enhanced by NADPH, and inhibited at 4°C. The cell‐free system, like intact cells, metabolized [3H]LTB4 to ω‐oxidized product rapidly and quantitatively at 37°C but was inactive at 4°C. Whole cells converted radiolabel to 20‐hydroxy (∼ 30% of product) and 20‐carboxy (∼ 70% of product) derivatives; the cell‐free system formed principally 20‐hydroxy‐[3H]LTB4. These products were less bioactive than LTB4. Nevertheless, metabolism of LTB4 played little role in limiting the cells' response to the ligand: neutrophils completed degranulation and became desensitized to LTB4 within 3–5 min of exposure. Within this time frame, they oxidized less than 30% of the stimulus, and the extracellular fluid of these neutrophil suspensions was fully capable of activating fresh cells. We conclude that neutrophils transmit bioactions of LTB4 via a specific receptor integrally associated with their plasmalemma and/or endoplasmic reticulum. They inactivate the stimulus via a particulate ω‐oxidase. At the level of the individual cell, receptor down‐regulation, rather than ligand metabolism, appears to limit functional responses such as degranulation.
Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet‐derived growth factor
Authors: Benitz William E. , Lessler Daniel S. , Coulson John D. , Bernfield Merton
Published: February 2005

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin‐Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet‐derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparinlike inhibitors of smooth muscle cell growth.
Effects of retinoids on human bronchial epithelial cells: Differential regulation of hyaluronate synthesis and keratin protein synthesis
Authors: Wu Reen , Wu Mary M. J.
Published: February 2005

Respiratory tract epithelia are one type of tissue targeted by vitamin A. In this study the effects of vitamin A and its analogs (retinoids) on human bronchial epithelial (HBE) cells have been investigated in a serum‐free hormone‐supplemented medium. This serum‐free medium, which was developed for the longterm cultivation of protease‐dissociated HBE cells, consists of Ham's F12 nutrient medium supplemented with insulin, transferrin, epidermal growth factor, hydrocortisone, cholera toxin, and bovine hypothalamus extract. Under these in vitro conditions, retinoids specifically stimulate the synthesis and secretion of hyaluronate (HA) and alter the pattern of synthesis of keratin proteins. In regard to HA, the degree of stimulation ranges from two‐fold to ten‐fold and is concentration dependent. In regard to keratin proteins, the most prominent effects of retinoids are inhibition of synthesis of the 48 kd and 50 kd keratin proteins (corresponding to cytokeratins 16 and 14, respectively, in the catalog of human cytokeratins; Moll et al., 1982) and stimulation of synthesis of the 40 kd and 52–54 kd proteins. The data indicate that retinoid effects on HA and keratin protein synthesis occur at different levels. The stimulation of HA synthesis occurs immediately after the addition of retinoid and cannot be prevented by pretreatment with actinomycin D, whereas the alterations in the pattern of keratin protein synthesis appear later and are inhibited by treatment with actinomycin D at or before the administration of retinoid. This study demonstrates that HBE cultures maintained in the serum‐free condition can serve as an in vitro model to elucidate the mechanisms of retinoid actions.
Heparin potentiation of 3T3‐adipocyte stimulated angiogenesis: Mechanisms of action on endothelial cells
Authors: Castellot John J. , Kambe Arbetta M. , Dobson Deborah E. , Spiegelman Bruce M.
Published: February 2005

We have examined the cellular mechanisms by which heparin potentiates the ability of 3T3‐adipocytes to stimulate the formation of new blood vessels. Both anticoagulant and non‐anticoagulant heparin species enhanced the angiogenic activity of adipocyte‐secreted products in the chick chorioallantoic membrane assay, indicating that the angiotropic effect of this glycosaminoglycan is independent of its effect on the coagulation cascade. Heparin alone was unable to produce a neovascular response. The ability of heparin to modulate three endothelial functions in vitro thought to be related to angiogenesis were examined: protease activity, motility, and mitogenesis. Heparin caused a 100% increase in the adipocyte‐induced stimulation of endothelial cell plasminogen activator activity and motility, but had no effect on proliferation. The enhancement of plasminogen activator and chemoattractant activities had a similar ED50 (1‐2 μg/ml) and optimum dose (10‐30 μg/ml). When we examined the direct effect of heparin on the activity of two distinct plasminogen activator enzymes–urokinase and tissue‐type–a dual action of heparin was observed: tissue‐type enzyme activity was stimulated 100% by heparin at 10 μg/ml, whereas urokinase activity was inhibited by 77% at this dose. These data suggest that heparin potentiates angiogenesis in vivo by stimulating endothelial cell plasminogen activator, motility, or both. Our results further suggest that for adipocyte‐induced blood vessel formation, in contrast to other angiogenesis systems, heparin does not appear to affect the mitogenic activity.
Fetuin: A serum component associated with rat sertoli and spermatogenic cells in coculture
Authors: Abdullah Munir , Crowell James A. , Tres Laura L. , Kierszenbaum Abraham L.
Published: February 2005

Cocultures of rat Sertoli‐spermatogenic cells plated in a culture medium supplemented with 10% fetal bovine serum for 6–12 h and then maintained in serum free, hormone/growth factor‐supplemented medium accumulated an acidic glycoprotein of molecular weight of 68,000 dalton (68 kD) and isoelectric point range of about 4.2–3.5. Anion exchange chromatography has allowed the partial purification of this protein, which consists of a major protein band of 68 kD and two minor, low molecular weight components. A rabbit antiserum raised against the 68 kD component also crossreacts with the two low molecular weight components, thus suggesting that these two minor components are antigenically related to the 68 kD protein. The 68 kD protein has been identified as fetuin, the major component of fetal bovine serum, based on similar molecular weight, isoelectric point, immunoreactivity and trypsin inhibitory activity. Labeling experiments with [14C]amino acid mixture show that 68 kD protein is not synthesized by cocultured rat Sertoli and spermatogenic cells. Immunocytochemistry and Western blot approaches carried out under various experimental conditions support the view that the fetuin‐68 kD protein is taken up from serum by both Sertoli cells and pachytene spermatocytes. Because fetuin (1) behaves as a carrier protein for growth factors, (2) has protease inhibitory activity, (3) is preferentially internalized by Sertoli cells and pachytene spermatocytes and (4) fetal bovine serum‐supplemented medium impairs spermatogenic cell viability, there is a need to further define appropriate conditions for optimizing long‐term viability and differentiation of spermatogenic cells in vitro.
Effects of bacterial lipopolysaccharide on protein synthesis in murine peritoneal macrophages: Relationship to activation for macrophage tumoricidal function
Authors: Hamilton Thomas A. , Jansen Marilyn M. , Somers Scott D. , Adams Dolph O.
Published: February 2005

Early biochemical events in the response of murine peritoneal macrophages to bacterial lipopolysaccharide (LPS) have been examined (i.e., 0–4 hr after initiation of treatment). At concentrations of 10 ng/ml or less, LPS stimulated the new or enhanced synthesis of a series of at least six polypeptides of 85, 80, 75, 65, 57, and 38 kD. This effect was dependent upon the lipid A moiety of LPS as lipid A itself could induce the changes and the effect of LPS could be blocked by inclusion of polymixin B sulfate in the culture medium. The effect was specific for LPS in that other endotoxin‐free agents known to alter macrophage physiology could not produce the same changes. The time course of LPS stimulation of macrophage protein synthesis was remarkable in that the synthesis of all six proteins was transient even in the continued presence of LPS, being first detected approximately 1 hr after exposure and no longer apparent by 8–10 hr after treatment was initiated. Furthermore, both pulse‐chase and cumulative radiolabeling studies indicated that at least two of the proteins (85 and 38 kD) were short‐lived and did not accumulate in LPS‐treated cells, suggesting the possibility that they participate in a regulatory rather than a functional role. Macrophage tumoricidal activation involves cooperation in response to two independent signals; interferon gamma and LPS. Pretreatment of macrophages with interferon gamma increased the sensitivity of macrophages to LPS‐stimulated protein synthesis by one to two orders of magnitude documenting such cooperativity in molecular terms. The LPS‐induced stimulation of specific protein synthesis could be reproduced by treatment of macrophages with heat killed Listeria monocytogenes, a gram‐positive, endotoxin‐negative bacterial stain which has been shown to substitute effectively for LPS in macrophage tumoricidal activation. Furthermore, reversible inhibition (i.e., treatment with cycloheximide) of protein synthesis during LPS treatment abrogated the acquisition of tumoricidal function. These results identify an early biochemical response to LPS which may be a necessary component of the intracellular transduction of signals which regulate macrophage functional development.
The 73 kilodalton heat shock cognate protein purified from rat brain contains nonesterified palmitic and stearic acids
Authors: Guidon Peter T. , Hightower Lawrence E.
Published: February 2005

A protein related to the 71 kilodalton inducible rat heat shock protein was purified to electrophoretic homogeneity in milligram amounts from brain tissue of nonheat‐stressed rats. The protein has been designated as a stress cognate protein based on previous studies and data presented herein that this protein cross‐reacted with a monoclonal antibody originally raised against the Drosophila 70 kilodalton heat shock protein. The purified protein had an apparent molecular mass of 73 kilodaltons when analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and an apparent mass of 150 kilodaltons as determined by nondissociative gel chromatography, suggesting that the purified protein is a homodimer. The purified protein had isoelectric points of 5.0 under nondissociative conditions and 5.6 when exposed to protein denaturants, suggesting loss of bound anionic molecules and/or net exposure of basic residues upon denaturation. Chloroform/methanol extraction of the purified protein and subsequent analyses by thin layer and gas‐liquid chromatography resulted in the identification of palmitic and stearic acids noncovalently bound to the protein. Approximately four molecules of fatty acids were bound per dimer with palmitic and stearic acids present in a one‐to‐one ratio. The purified protein did not bind exogenously added radio‐active palmitate, indicating that the fatty acid‐binding sites of the cognate protein were fully occupied and that the associated fatty acids were too tightly bound to exchange readily. The possible significance of the fatty acids associated with the 73 kilodalton stress cognate protein is discussed.
Comparative studies of the binding and growth‐supportive ability of mammalian transferrins in human cells
Authors: Penhallow Robert C. , Brown‐Mason Anne , Woodworth Robert C.
Published: February 2005

The ability of human‐derived cells in culture to bind, remove iron from, and grow in the presence of transferrins (Tf) isolated from the sera of species commonly included in tissue culture medium was investigated. Kinetic studies on HeLa cells reveal apparent first‐order association rate constants of 0.43 min−1 for human Tf and 0.15 min−1 for equine Tf. Labeled chicken ovo‐Tf and fetal bovine Tf were not recognized by the HeLa cells. Competition experiments with HeLa cells that use either isolated Tf or parent serum confirm these findings. Equilibrium binding experiments performed on HeLa cells at 37°C in the presence of 2,4‐dinitrophenol to prevent iron removal indicate 1 × 106 Tf bound/cell with a dissociation constant (K'D) of 28 nM for human Tf and 182 nM for equine Tf. Equilibrium binding performed at 0°C to prevent endocytosis reveals 4.1–6.7 × 105 Tf binding sites/cell with a K'D of 8.3 nM for human Tf and 41.5 nM for equine Tf. Parallel experiments in normal human diploid fibroblast‐like MRC‐5 cells indicate expression of 0.82–2.78 × 105 Tf binding sites/cell with a K'D of 8.2 nM for human and 39.1 nM for equine Tf. Thus, the results of equilibrium binding studies of a more differentiated cell type are consistent with those found for HeLa cells. Fetal bovine Tf was found to compete weakly with labeled human Tf for human receptor on HeLa cells in a soluble receptor assay, with an approximately 500‐fold excess needed to reduce binding to half maximal. Iron uptake experiments show an iron donating hierarchy where human > horse > calf, suggesting that the rate of iron uptake depends on the affinity of receptor for transferrin. Growth experiments involving HeLa cells in chemically defined serum‐free medium demonstrate that bovine Tf will support growth as well as human Tf, but at concentrations much higher than are required of human Tf.