bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular processing of residualizing labels in different cell types in vitro
Authors: Lucas Karen L. , Baynes John W. , Thorpe Suzanne R.
Published: February 2005

In previous autoradiographic studies on the sites of catabolism of rat serum albumin (RSA) in the rat, fibroblasts in skin and muscle were shown to accumulate degradation product from RSA labeled with the residualizing label dilactitol‐125I‐tyramine (125I‐DLT) (Strobel et al., 1986 J. Biol. Chem., 261:7989—7994). Residualizing labels remain at the cellular site of degradation of the carrier protein because of their size, hydrophilicity, and resistance to lysosomal hydrolases. This study was designed to evaluate whether fibroblasts might retain labeled degradation products more efficiently than other cell types. The uptake of 125I‐DLT‐RSA and release of its degradation products and of a second non‐biodegradable probe, fluorescein isothiocyanate (FITC)‐dextran, were studied in fibroblasts, endothelial cells, and macrophages, all cell types previously implicated in the catabolism of albumin in vivo. The rates of uptake of labeled protein and dextran were comparable in all cell types and consistent with fluid phase endocytosis. The rate of release of both intact protein (30—35% of total radioactivity released) and radioactively labeled degradation products followed similar kinetics and had half‐lives ranging from 26 to 37 hr. The rate of release of FITC‐dextran was slower than that of radioactivity, with a half‐life of 42—125 hr. Thus, although there were differences between the rates of release of the fluorescent and radioactive materials in vitro, there were no significant differences in the disposition of protein‐derived catabolites among these three cell types.
Osmotic and motional properties of intracellular water as influenced by osmotic swelling and shrinkage of Xenopus Oocytes
Authors: Cameron Ivan L. , Merta Phillip , Fullerton Gary D.
Published: February 2005

Experiments were done on fully grown Xenopus oocytes to determine the extent and the properties of cellular water of hydration. The studies involved the osmotic shrinking and swelling of the oocytes under known osmotic pressure as well as proton NMR spectral, titration, and free induction decay analyses. Studies were done both on whole oocytes and on subcellular fractions. The results show that little if any of the oocyte water in situ has the motional or osmotic properties expected of pure "bulk" water. Four distinct water of hydration compartments were found and defined on the basis of distinct hydrogen bounding mechanisms. Some of the water in yolk platelets was found not to be in fast exchange with other water compartments. Osmotic shrinkage of oocytes caused an adaptive decrease in the bound water of hydration compartments. This osmotically induced decrease is attributed to decreased surface area available for the hydrogen bounding of water molecules on cellular proteins.
Reduction of DNA primase activity in aging but still proliferating cells
Authors: Collins James M. , Chu Annie K.
Published: February 2005

The basis of the well‐known decline in cell proliferation with increasing passage number of human diploid fibroblast‐like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase α bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165‐173, 1985]. In the present study, the declining percentages of S‐phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase α and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase‐digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase α and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrase in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.
Expression of externally‐disposed heparin/heparan sulfate binding sites by uterine epithelial cells
Authors: Wilson Oswald , Jacobs Andrew L. , Stewart Shelley , Carson Daniel D.
Published: February 2005

A class of high‐affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time‐dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]‐Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 × 106 sites per cell. Approximately 9–14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo‐uterine and epithelial‐basal lamina interactions.
Regulation of hexose transport in respiration deficient hamster lung fibroblasts
Authors: Germinario Ralph J. , Andrejchyshyn Susan , Kristof Arnold , Chang Zully , Oliveira Maureen , Citrynbaum Leo
Published: February 2005

The transport of [3H]2‐deoxy‐D‐glucose (2DG) and [3H]3‐O‐methyl‐D‐glucose (3‐OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 ± 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 ± 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 ±.6 vs. 3.0 ±.13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I‐insulin binding to cells, and insulin‐stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1‐50 μg/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cyc‐loheximide concentrations resulted in decreased 2DG transport, while higher concentrations (≥ 1 μg/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 μg/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.
Proliferation‐associated changes of Ca2+ transport in myeloid leukemic cell lines
Authors: Rephaeli Ada , Aviram Adina , Rabizadeh Ester , Shaklai Mati
Published: February 2005

Proliferation‐associated changes in calcium metabolism were investigated employing the promyelocytic HL‐60 and monoblastic U‐937 cell lines. The cells were stimulated to proliferate employing mitogenic factors as follows. 1) Transferrin or insulin: HL‐60 cells were adjusted for growth in serum‐free medium, and 24 h prior to the experiment, the cells were deprived of transferrin or insulin. The re‐addition of either one of them stimulated cell proliferation as was evident by increased [3H]‐tymidine incorporation activity. Cell proliferation was associated with an enhanced Ca2+ influx rate, measured by 45Ca2+ uptake activity. 2) Granulocyte‐monocyte colony‐stimulating factor (GM‐CSF): addition of GM‐CSF to proliferating or quiescent HL‐60 cells resulted in increased cell proliferation, which was also accompanied by increased rate of Ca2+ influx. 3) Serum: HL‐60 and U‐937 were grown for 24 h in serum‐depleted medium. Re‐addition of serum to the cells was not associated with immediate or delayed change in calcium influx rate but rather with an immediate increase in the cytosolic free calcium concentration, measured employing the fluorescent probe, fura‐2AM. This increase was independent of extracellular calcium, unaffected by verapamil, diltiazem, and lanthanum, and associated with enhanced 45Ca2+ efflux. Thus, in all three cases evoked cell proliferation was accompanied by quantitative changes in Ca2+ metabolism. While the transferrin‐, insulin‐, and GM‐CSF‐stimulated cell proliferation was accompanied by delayed increases in 45Ca2+ influx, the serumstimulated cell proliferation was accompanied by an immediate elevation of free cytosolic Ca2+.
Effect of second‐messenger modulators in K‐562 cell differentiation: Dual action of calcium/phospholipid‐dependent protein kinase in the process of differentiation
Authors: Yumoto Yoshikazu , Tashima Masaro , Kato Yoshiki , Ueda Takeo , Okuda Tetsuya , Ogawa Kazuei , Sawada Hiroyoshi
Published: February 2005

We investigated the roles of second messengers in K‐562 cell differentiation induced by either commitment‐inducing agents (Ara‐C, thymidine), or a noncom‐mitment‐inducing agent (hemin). Cell differentiation induced by both types of agents was inhibited by dbc‐AMP, staurosporine, and H‐7. In contrast, OAG enhanced hemin‐induced cell differentiation, but it inhibited that due to Ara‐C or thymidine. When K‐562 cells were incubated with 4 x 10−6M hemin or 2 x 10−7M Ara‐C for 2 days, an increase of ϵ‐mRNA occurred. The addition of cycloheximide (1 μg/ml) completely blocked this change, suggesting that de novo protein synthesis was necessary for the increase of ϵ‐mRNA. Simultaneous treatment with Ara‐C and cycloheximide for 2 days did not block either the increase of ϵ‐mRNA or that of benzidine‐positive cells, which were measured after 5 days of further incubation without additives. This suggested that the process of Ara‐C‐induced K‐562 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step, and that the commitment step was at least partly resistant to cycloheximide. We investigated the roles of second messengers in each step. Our results suggested that PKC may act as a negative regulator of commitment step and as a positive regulator of the phenotypic expression. This may explain the differing effects of OAG on hemin‐ and Ara‐C‐induced K‐562 cell differentiation.
Length‐dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle
Authors: Moorf Russell L. , Persechini Anthony
Published: February 2005

The effect of changes in muscle length on post‐tetanic isometric twitch tension potentiation and myosin P‐light chain phosphorylation‐was studied at 23°C in the mouse extensor digitorum longus muscle. The length‐tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L0. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L0 to a maximum of 0.3 at 1.2 L0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post‐tetanus. P‐light chain phosphorylation remains constant between 30 s and 3 min post‐tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P‐light chain phosphor‐ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L0 is 35% less than that obtained under the same conditions at L0. Thus, contraction‐induced phosphorylation of P‐light chain decreases with increased muscle length and post‐tetanic potentiation at a constant level of P‐light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca2+ ion transient as the muscle is lengthened.
p52 induction by cytochalasin D in rat kidney fibroblasts: Homologies between p52 and plasminogen activator inhibitor type‐1
Authors: Higgins Paul J. , Ryan Michael P. , Zeheb Ron , Gelehrter Thomas D. , Chaudhari Panna
Published: February 2005

Normal rat kidney (NRK) fibroblasts respond to the cell shape‐modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52‐kDa substrate‐associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43‐kDa “core” protein (p43), four 50‐kDa species (p50‐0,1,2,3), and at least seven distinct pl variants of the mature 52‐kDa protein. A threshold of 2–4 μM CD was found to be necessary to augment p52 deposition into both the secreted protein‐ and saponin‐resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD‐treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type‐1 (rPAI‐1) and to PAI‐1‐like proteins of other species by comparative immunoprecipitation, 2‐D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI‐1 cytoplasmic mRNA abundance, using the rPAI‐1 cDNA probe pSS1‐3, revealed an induction of rPAI‐1 mRNA in NRK/CD cells which paralleled the increased protein production. CD‐augmented p52(rPAI‐1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape‐associated metabolic processes and concomitant RNA synthesis.
Regulation of hexose transport in L8 myocytes by glucose: Possible sites of interaction
Authors: Wertheimer Efrat , Sasson Shlomo , Cerasi Erol
Published: February 2005

Previous work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down‐regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827–;16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2‐deoxy‐D‐glucose (dGlc) uptake by 40% within 3 h. Cyc‐loheximide (1 μM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 μM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up‐regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down‐regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady‐state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non‐glycosylated protein involved in the translocation or activation of glucose transporters.
Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study
Authors: Jeffrey John J. , Roswit William T. , Ehlich Lynn S.
Published: February 2005

An enzyme‐linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000‐fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross‐react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10−6M, and that of medroxyprogesterone acetate was 1 x 10−7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten‐fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.
Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice
Authors: Tennenbaum Tamar , Yuspa Stuart H. , Kapitulnik Jaime
Published: February 2005

The proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (<0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture med um from 1 to 5 mM stimulated the proliferation of newborn mouse (1–3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2–3‐fold higher in the magnesium‐enriched cultures. Exposure to high magnesium caused 3–4‐fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10–15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2+ M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)‐induced differentiation.
Cystine and dibasic amino acid uptake by opossum kidney cells
Authors: States Beatrice , Segal Stanton
Published: February 2005

The characteristics of the uptake of L‐cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC‐PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high‐affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cysteine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.
Transforming growth factor‐β1 localization in normal and psoriatic epidermal keratinocytes in situ
Authors: Kane Cynthia J. M. , Hanawalt Philip C. , Knapp A. Merrill , Mansbridge Jonathan N.
Published: February 2005

Transforming growth factor‐β1 (TGFβ1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGFβ1‐specific polyclonal antibodies (anti‐LC and anti‐CC) to determine the presence of TGFβ1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti‐TGFβ1 staining was observed primarily in the epidermis. Anti‐LC TGFβ1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti‐CC TGFβ1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti‐LC and anti‐CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGFβ1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGFβ1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGFβ1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a Physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGFβ1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.
Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: Possible role as differentiation‐signal
Authors: Beit‐Or A. , Nevo Z. , Kalina M. , Eilam Y.
Published: February 2005

Cell‐ and matrix‐related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen‐storing pools. The rate of proteoglycan synthesis gradually increased. Age‐related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura‐2‐loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5‐day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34‐day cultures. On the basis of the present data and the previous results, which showed that cartilage‐derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i, caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady‐state levels of [Ca2+]i.
Astrocytes induce neural microvascular endothelial cells to form capillary‐like structures in vitro
Authors: Laterra John , Guerin Christopher , Goldstein Gary W.
Published: February 2005

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial‐endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillarylike structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72‐96 hours following the addition of BRE cells to 1‐day‐old astroglial cultures. The extent of BRE reorganization was quantitated by computer‐assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6‐conditioned media and C6‐endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6‐endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial‐astroglial interface of capillary‐like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary‐like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.
Synthesis of basic fibroblast growth factor upon differentiation of rat mammary epithelial to myoepithelial‐like cells in culture
Authors: Barraclough Roger , Fernig David G. , Rudland Philip S. , Smith John A.
Published: February 2005

Acidic fibroblast growth factor (aFGF) mRNA was detected in a rat mammary fibroblastic cell line, but not in rat mammary epithelial cell lines or myoepithelial‐like cell lines. Basic FGF (bFGF) mRNA was detected in both the fibroblasts and the myoepithelial‐like cells, but was absent from the epithelial cells. A series of cell lines representing stages in the differentiation pathway of epithelial cells to a myoepithelial‐like morphology showed an increase in the amount of bFGF mRNA and activity present and the FGF from the myoepithelial‐like rat mammary 29 cells was able to displace [125I]‐bFGF specifically bound to rat mammary fibroblasts. FGF activity was also present in an extract of rat mammary gland. Analysis of cell extracts and conditioned medium indicated that FGF activity was cell‐associated. The cell‐associated bFGF was resistant to degradation by trypsin. Extraction of myoepithelial‐like cells with Triton X‐100 and 2 M NaCl showed that 50–65% of the cell‐associated bFGF was in a detergent‐resistant but 2 M NaCl− labile structure. Thus, the synthesis of bFGF is developmentally regulated in rat mammary cell lines, and at least 50% is present in the extracellular matrix.
Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics
Authors: Zaitsu Hirokazu , Serrero Ginette
Published: February 2005

Crude Pedersen fetuin, derivea from fetal bovine serum, contains adipogenic activity, Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S‐300, the majority of adipogenic activity eluted in two distinct peaks, F1 (molecular weight greater than 669 kDa) and Fii (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, Fiii (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. F1 has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. Fii has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein α2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.
Synergism between gangliosides and basic fibroblastic growth factor in favouring survival, growth, and motility of capillary endothelium
Authors: Alessandri G. , De Cristan G. , Cappa A. P. M. , Gullino P. M. , Morbidelli L. , Ziche M.
Published: February 2005

The experiments reported were motivated by the observation that in vivo gangli‐osides promoted angiogenesis when the dose of the angiogenic factor was too low to be effective (Ziche et al.: Laboratory Investigation 61:629–634, 1989). As an approach to understanding the mechanism of this modulatory effect, we analysed the influence that gangliosides have on survival, growth, and migration of capillary endothelium when and angiogenesis factor like basic fibroblast growth factor (bFGF) was present in the culture medium. Clones of bovine capillary endothelium were cultivated in media unable to sustain survival over a 72 h period. With this experimental approach, cell survival was evaluated after addition of either bFGF or gangliosides or both to the medium. The Boyden chamber procedure was utilized to measure the influence of bFGF or gangliosides on cell mobilization across a micropore filter. Low doses of both molecules, ineffective when added singly to the culture media, improved all three parameters when added in combination. A synergic effect between bFGF and the gangliosides (GM1, GD1b, GT1b) was observed for the improvement of survival or growth and for the acceleration of endothelial cell migration. The removal of sialic acid from the ganglioside molecule prevented any effect on all three parameters. The addition of sialic acid alone to cultures was also totally ineffective. In the aduit organism most angiogenic events occur under conditions of tissue damage. The synergism between gangliosides and bFGF can be interpreted as the initial phase of a process for which endothelial cell survival is the indispensable first step in the formation of a new vascular network.
PDGF‐stimulated fibroblast proliferation is enhanced synergistically by receptor‐recognized α2‐Macroglobulin
Authors: Bonner James C. , Badgett Annette , Osornio‐Vargas Alvaro R. , Hoffman Maureane , Brody Arnold R.
Published: February 2005

α‐Macroglobulins derived from plasma or secreted by macrophages are plateletderived growth factor (PDGF) binding proteins that compete with cell‐surface receptors on fibroblasts for PDGF binding. α2‐Macroglobulin (α2M) derived from bovine plasma was tested for its ability to modulate the PDGF‐induced proliferation of primary passage rat lung fibroblasts (RLFs) and a human skin fibroblast cell line (CRL 1508). Fibroblasts were grown in 10% fetal bovine serum (FBS) for 24 hr, then washed with serum‐free medium before adding serum‐free defined medium (SFDM) containing insulin and transferrin. To this medium were added varying concentrations of human plasma‐derived AB‐PDGF and α2M, alone or in combination. Receptor‐recognized α2M was prepared by treatment with methylamine. Both native α2M and the α2M‐methylamine (α2M‐MA) were tested for growth promoting activity in the absence or presence of PDGF. After 3 days, a concentration‐dependent growth curve of fibroblast proliferation was demonstrated for PDGF alone, with near maximal stimulation reached at 15–20 ng/ml PDGF. α2M and α2M‐MA alone had no effect on cell proliferation. However, α2M‐MA concentrations above 32 μg/ml synergistically enhanced PDGF‐stimulated proliferation >100% in the presence of 15 ng/ml PDGF. Native α2M enhanced PDGF‐stimulated growth 80–100% above PDGF controls only at low concentrations (32–64 μg/ml α2M). High concentrations of native α2M (128–256 μg/ml) either had no effect on growth or were inhibitory to PDGF‐stimulated growth, depending on the cell type tested. Rat lung fibroblasts were shown to secrete a factor(s) that inhibited the trypsin‐binding capacity of native α2M. We further demonstrated that early passage RLFs possess specific cell‐surface receptors for [125I]‐PDGF and [125I]‐α2M‐MA, and preincubation of RLFs with α2M‐MA increased the specific binding of [125I]‐PDGF to the cell surface of these fibroblasts. Considered together, these data support the view that receptor‐recognized α2M synergistically enhances the proliferative capacity of PDGF. We postulate that receptor‐recognized αMs enhance PDGF‐stimulated growth by increasing the local concentration of PDGF at the cell surface, where the PDGF could be released in close proximity to its own receptors.
Transcellular processing of disulfide‐and thioether‐linked peroxidase‐polylysine conjugates in cultured MDCK epithelial cells
Authors: Wan Jiansheng , Persiani Stefano , Shen Wei‐Chiang
Published: February 2005

Horseradish peroxidase (HRP) was conjugated to nondegradable polycationic poly(D‐lysine) (PDL) through either a thioether (HRP‐S‐PDL) or a disulfide (HRP‐SS‐PDL) linkage. The binding and transcytosis of these conjugates was studied in Madin‐Darby canine kidney (MDCK) cell monolayers grown on 3‐μm microporous polycarbonate filters. Conjugation of HRP to PDL with both linkages markedly increased the binding of this protein onto the cell monolayers. However, an enhancement of the transcellular transport of HRP in both apical‐to‐basal and basal‐to‐apical directions was observed only in HRP‐SS‐PDL, but not in HRP‐S‐PDL. HRP‐SS‐PDL transport was inhibited by colchicine and by 4oC incubation. The transport of 14C‐sucrose was not affected by the presence of conjugates. These results indicate that the transport of the conjugate across the cell monolayers was due to a transcellular process rather than to any leakage of the cell junction caused by polycations. The disulfide linkage between HRP and PDL was cleaved rapidly at the basal and, to a lesser extent, at the apical surface of the cell. Neuraminidase treatment decreased the binding of the conjugates onto the cell surface, but did not decrease the transcellular transport, suggesting that not all surface‐bound conjugates were available for transcytosis. These results demonstrate that disulfide linkages can be cleaved during transcytosis in MDCK cells. The cleavage, however, occurs mostly at the binding site on the cell surface, which may prevent the cellular uptake of the intact conjugate.
Thrombospondin gene expression is associated with mitogenesis in 3T3 cells: Induction by basic fibroblast growth factor
Authors: Donoviel Dorit B. , Amacher Sharon L. , Judge Kevin W. , Bornstein Paul
Published: February 2005

Growth factor‐depleted Swiss 3T3 cells responded to basic fibroblast growth factor (bFGF) with a burst of mitogenesis and with a rapid and marked increase in thrombospondin (TS) mRNA levels. mRNA levels for the α1 chain of type I collagen and for fibronectin were unaffected. At early times following stimulation (0–2 h), “superinduction” of TS mRNA by inhibition of protein synthesis with cycloheximide was not observed and the increase in TS mRNA could be attributed primarily to an increase in transcription rate of the TS gene. However, at later times (4–8 h) the combination of cycloheximide and bFGF superinduced TS mRNA levels, suggesting the existence of a labile inhibitor of transcription or a short‐lived RNase that might be produced in response to prolonged treatment with bFGF. In contrast to its stimulatory effect on 3T3 cells, bFGF did not stimulate the proliferation of mouse muscle BC3H1 cells nor did it cause an increase in TS mRNA levels, but BC3H1 cells do respond to bFGF by inhibition of myogenic differentiation. We propose, on the basis of these and other findings, that TS facilitates the progression of some anchorage‐dependent cells through the cell cycle.
A factor contained in plasma is required for IGF binding protein‐1 to potentiate the effect of IGF‐I on smooth muscle cell DNA synthesis
Authors: Clemmons D. R. , Gardner L. I.
Published: February 2005

One of the forms of the insulin‐like growth factor (IGF) binding proteins present in human amniotic fluid has been shown to potentiate the growth‐promoting effect of IGF‐I markedly. This study was undertaken to determine the cellular and hormonal factors that modulate this potentiation and to determine whether this protein would potentiate the effects of other mitogens. Although the combination of the IGFBP‐1 (20 ng/ml) and IGF‐I (10 ng/ml) induced a five‐ to sixfold increase in DNA synthesis compared with IGF‐I alone, this response required the simultaneous addition of IGF‐I with 0.1% platelet‐poor plasma (PPP). If PPP was omitted from the incubation medium, no increase above the effect that was obtained with IGF‐I alone was noted. Substitution of cerebrospinal fluid (CSF) for PPP permitted a full mitogenic response, although substitution with amniotic fluid resulted in no enhancement. The factor contained in PPP was heat and acid stable. If the binding protein was co‐incubated with fibroblast growth factor (FGF), platelet‐derived growth factor (PDGF), or epidermal growth factor (EGF), a slight inhibition of the cellular response to each of these factors was detected. Co‐incubation of IGF‐I with the IGF‐binding protein plus these other peptide growth factors resulted in no further enhancement of DNA synthesis above the level observed with IGF‐I and the binding protein alone. Likewise, addition of plasma proteins such as transferrin or albumin did not result in a further enhancement of the DNA synthesis response to IGF‐I plus binding protein, and these proteins could not substitute for PPP or IGFBP‐1. Transient exposure of the cultures (2 hr) to the binding protein plus IGF‐I resulted in a submaximal DNA synthesis response, and the binding protein had to be present continuously to achieve a maximal effect. These studies indicate that a factor contained in plasma and CSF is required for a maximal cellular response to IGFBP‐1 plus IGF‐I, and this factor does not appear to be a well‐defined mitogen.
Evidence for involvement of a nuclear envelope‐associated RNA helicase activity in nucleocytoplasmic RNA transport
Authors: Schröder Heinz C. , Ugarkovic Durdica , Langen Peter , Bachmann Michael , Dorn August , Kuchino Yoshiyuki , Müller Werner E.G.
Published: February 2005

It seems well established that translocation of at least some mRNAs through the nuclear pore is (1) an energy‐dependent process, and (2) dependent on the presence of the poly(A) segment attached to most mRNA species. We describe that RNA helicase (RNA duplex unwindase) activity is present in a nuclear envelope (NE) preparation, which also appears to be involved in nucleocytoplasmic RNA transport. This activity unwinds RNA : RNA hybrids. The helicase has a pH optimum of 7.5 and a temperature optimum of 30°C. Applying the sealed NE vesicle system, it was shown that duplex RNA species are readily released from the vesicles in an unidirectional manner, in contrast to single‐stranded RNA, which is much slower transported into the extravesicular space. Attachment of a poly(A) segment to the RNA duplex additionally increases the efflux rate of this RNA. Efflux of duplex RNA but not efflux of single‐stranded RNA was strongly inhibited by formycin B 5′‐triphosphate. Our results suggest that, besides poly(A), duplex structures, if present in a given RNA, modulate and control the export of RNA.
Stromal influences on transformation of human mammary epithelial cells overexpressing c‐myc and SV40T
Authors: Valverius Eva M. , Ciardiello Fortunato , Heldin Nils‐Erik , Blondel Bruno , Merlo Giorgio , Smith Gilbert , Stampfer Martha R. , Lippman Marc E. , Dickson Robert B. , Salomon David S.
Published: February 2005

The proto‐oncogene c‐myc and the oncogene SV40T, both of which have been implicated in the process of cellular immortalization in vitro, have been introduced via amphotropic retroviral expression vectors into the human mammary epithelial cell (HMEC) line 184A1N4 (A1N4). Two stable cell lines were established by growth in selective medium and were found to overexpress either c‐myc (A1N4‐myc) or SV40T antigen (A1N4‐T). Neither the A1N4, A1N4‐myc, or A1N4‐T cells will grow in soft agar or form tumors in nude mice. However, A1N4‐T or A1N4‐myc cells, but not the parental A1N4 cells, form colonies in soft agar in response to either epidermal growth factor (EGF), transforming growth factor α (TGFα), or basic fibroblast growth factor (bFGF). Like EGF and TGFα, bFGF is moderately mitogenic for the anchorage‐dependent growth (ADG) of all three cell lines. Further, co‐cultivation of A1N4‐T or A1N4‐myc cells with primary diploid mammary fibroblasts can also induce the anchorage‐independent growth (AIG) and stimulate the ADG of A1N4‐T or A1N4‐myc. In addition, conditioned medium obtained from these mammary fibroblasts also stimulated the AIG of the A1N4‐T and A1N4‐myc cells and was found to contain immunoreactive TGFα and bioactive FGF. The mammary fibroblasts express specific mRNA transcripts for bFGF and acidic FGF (aFGF). These results suggest that growth factors such as TFGα or FGF, which may be derived from the adjacent mammary stroma, might influence in a paracrine manner the phenotypic characteristics of a population of human mammary epithelial cells toward transformation.
The Na+,K+,2Cl−‐cotransport system in HeLa cells: Aspects of its physiological regulation
Authors: Kort Jens J. , Koch Gebhard
Published: February 2005

We have previously reported on the biochemical properties of a Na+,K+,2Cl−‐cotransport in HeLa cells and here we deal with aspects of its physiological regulation. Na+,K+,2Cl−‐cotransport in HeLa cells was studied by 86Rb+ influx and 86Rb+/22Na+ efflux measurements. The effects of rat atrial natriuretic peptide (ANP), isoproterenol, and amino acids on 86Rb+ flux, mediated by the bumet‐anide‐sensitive Na+, K+, 2Cl−‐cotransport system and the ouabain‐sensitive Na+/K+‐pump, were investigated. ANP reduced bumetanide‐sensitive 86Rb+ influx under isotonic as well as under hypertonic conditions. Similar decrease of bumetanide‐sensitive 86Rb+ influx was observed in the presence of 8‐bromo‐cGMP, while neither isoproterenol as a β‐receptor agonist nor 8‐bromo‐cAMP‐could alter bumetanide‐sensitive 86Rb+ influx. Furthermore, efflux of 86Rb+ and 22Na+ was greatly reduced in the presence of bumetanide and ANP. Together with our recent findings, showing functionally active, high affinity receptors for ANP on HeLa cells (Kort and Koch, Biochim. Biophys. Res. Commun. 168:148–154, 1990), this study indicates that ANP participates in the regulation of the Na+, K+, 2Cl−‐cotransport system in HeLa cells. Further measurements revealed that amino acids as present in the growth medium (Joklik's minimal essential medium) and the amino acid derivative α‐methyl‐aminoisobutyric acid (metAlB, 1 and 5 mM, respectively) also reduced Na+, K+, 2Cl−‐cotransport‐mediated 86Rb+ uptake and diminished the stimulatory effect of hypertonicity on the cotransporter. In addition, the Na+/K+‐pump was markedly stimulated in the presence of amino acids, while neither ANP and 8‐Br‐cGMP nor isoproterenol and 8‐Br‐cAMP had a significant effect on the activity of the Na+/K+‐pump.
Extracellular matrix derived from Trypanosoma cruzi infected endothelial cells directs phenotypic expression
Authors: Morris Stephen A. , Wittner Murray , Weiss Louis , Hatcher Victor B. , Tanowitz Herbert B. , Bilezikian John P. , Gordon Portia B.
Published: February 2005

Infection of confluent human umbilical vein endothelial cells by the parasite Trypanosoma cruzi results in the appearance of an altered heparan sulfate proteoglycan (HSPG) isolated from the extracellular matrix of infected endothelial cells (ECMi). HSPG from ECMi differed from HSPG obtained from the extracellular matrix of uninfected endothelial cells (ECMu) by virtue of an 8–10‐fold increase in sulfation and a different elution pattern using DEAE Sepharose chromatography. Analysis of the HSPG that binds to acidic fibroblast growth factor (aFGF) revealed that infection increased the proportion of HSPG that binds to aFGF by 35%. Heparitinase and alkaline borohydride treatment of aFGF‐binding HSPG and chromatographic resolution on Sepharose CL4B column revealed an infection‐associated 10‐fold increase in sulfation of the GAG side chain with no significant change in the migration of the core protein. In addition, the aFGF binding HSPG isolated from ECMi demonstrated a markedly attenuated synergistic mitogenic activity with aFGF in a cell proliferation assay. All of the infection associated changes in HSPG could be demonstrated in HSPG obtained from uninfected endothelial cell cultures grown on ECMi. Hence, the ECMi is associated with signals capable of modulating the ECM associated metabolism of uninfected endothelial cells. This facility of ECMi was also shown to extend to patterns of Gs protein synthesis as revealed by Western blot analysis. The observation that the ECM produced by infected endothelial cells can direct the synthetic patterns of uninfected endothelial cells in a manner uniquely observed in infected endothe‐lial cells suggests a plausible pathway by which infection of only a few cells can ultimately result in the coordinate responses of neighboring uninfected cells.