bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phagocytosis in Acanthamoeba: I. A mannose receptor is responsible for the binding and phagocytosis of yeast
Authors: Allen Philip G. , Dawidowicz Eliezar A. 
Published: February 2005

AbstractWe have examined the initial events in phagocytosis by Acanthamoebe castellanii in order to understand this process at the molecular level and have determined that phagocytosis in this organism is mediated by a receptor which recognizes mannose‐ricn elements in the particle to be phagocytosed. We demonstrate that the binding and internalization of yeast particles can be inhibited by the sugars D(+)‐mannose and D(−)‐fructose in a stereospecific, concentration‐dependent manner. This inhibition is specific; these sugars did not inhibit the uptake of latex beads by this organism. Using mannosylated neoglycoproteins, which are much more potent inhibitors of particle binding as compared with the free sugar, we demonstrate the presence of a receptor on the amoeba cell surface which is necessary for the binding of yeast as the initial event of phagocytosis. The Acanthamoeba mannose receptor also appears to be able to mediate the delivery of soluble mannose‐rich molecules to a degradative compartment such as the lysosome. Knowledge of this receptor will allow a better understanding of the molecular events of phagocytosis.
Differential modes of action of fibronectin and epidermal growth factor on rabbit corneal epithelial migration
Authors: Nishida Teruo , Nakamura Masatsugu , Mishima Hiroshi , Otori Toshifumi 
Published: February 2005

AbstractIn order to clarify the roles of fibronectin (FN) and epidermal growth factor (EGF) in corneal wound healing, we cultured blocks of excised rabbit cornea for 24 hours in media containing one of these agents, then measured the length of the path of the epithelial layer that had migrated down the side of the block. Both FN and EGF stimulated epithelial migration significantly in a dose‐dependent fashion. Responses to EGF involved a time ag of at least 12 hours before stimulation could be observed, but there was no lag‐time for FN‐stimulated migration. FN was maximally effective only if it was continuously present. In contrast, exposure to EGF for 6 hours did not stimulate epithelial migration, but exposure for 9 hours resulted in the same stimulatory effects as were observed after 24 hours' continuous exposure. Anti‐FN antibody inhibited the FN‐ and EGF‐stimulated migration of corneal epithelium. But anti‐EGF antibody inhibited only EGF‐stimulated migration and had no effect on FN‐stimulated migration. These results indicate that, unlike FN, EGF need not be present, once the epithelial cells have recognized its signal. Furthermore, the stimulatory effect of EGF depended on FN, while that of FN was independent of EGF. The effects of EGF on migration of corneal epithelium may, therefore, be mediated by FN.
Demonstration of a direct role for myosin light chain kinase in fibroblast‐populated collagen lattice contraction
Authors: Ehrlich H. Paul , Rockwell W. Bradford , Cornwell Trudy L. , Rajaratnam Joseph B. M. 
Published: February 2005

AbstractMixing feed fibroblasts with soluble collagen and serum‐supplemented culture medium at 37°C results in the entrapment of cells within the polymerizing collagen matrix. This cellular‐collagen complex is referred to as a fibroblast‐populated collagen lattice (FPCL). In time, this FPCL undergoes a reduction in size called lattice contraction. The proposed mechanism for lattice contraction is cellular force produced by cytoplasmic microfilaments which organize collagen fibrils compacting the matrix. When the regulatory subunits of myosin, myosin light chains, are phosphorylated by myosin light chain kinase (MLCK), myosin ATPase activity is increased and actin‐myosin dynamic filament sliding occurs. Elevated levels of myosin ATPase are required for maximal lattice contraction. Cholera toxin inhibits lattice contraction by increasing intracellular levels of cAMP. It is proposed that increased cytoplasmic concentrations of cAMP promote phosphorylation of MLCK, the enzyme important for maximizing myosin ATPase activity. Phosphorylating MLCK in vitro inhibits activity by decreasing its sensitivity to calcium‐calmodulin complex. A decrease in MLCK activity would result in lower levels of myosin ATPase activity. MLCK, purified from turkey gizzard, was subjected to limited proteolytic digestion to produce calmodulin‐independent‐MLCK. The partially digested kinase does not require calcium‐calmodulin for activation. Independent‐MLCK is not subject to inhibition by phosphorylation. The electroporetic inoculation of independent‐MLCK into fibroblasts before FPCL manufacture produced enhanced lattice contraction. Lattice contraction, in the presence of cholera toxin, was restored to normal levels by the prior electroporetic introduction of independent‐MLCK. These findings support the hypothesis that increases in cAMP hinder lattice contraction by a mechanism involving inhibition of MLCK and myosin ATPase.
Macrovascular and microvascular endothelium during long‐term hypoxia: Alterations in cell growth, monolayer permeability, and cell surface coagulant properties
Authors: Shreeniwas R. , Ogawa S. , Cozzolino F. , Torcia G. , Braunstein N. , Butura C. , Brett J. , Lieberman H. B. , Furie M. B. , Joseph‐Silverstein J. , Stern D. 
Published: February 2005

AbstractIn bovine aortic or capillary endothelial cells (ECs) incubated under hypoxic conditions, cell growth was slowed in a dose‐dependent manner at lower oxygen concentrations, as progression into S phase from G1 was inhibited, concomitant with decreased thymidine kinase activity. Monolayers grown to confluence in ambient air, wounded, and then transferred to hypoxia showed decreased ability to repair the wound, as a result of both decreased motility and cell division. Hypoxic ECs demonstrated a ≈3‐fold increase in the total number of high‐affinity fibroblast growth factor receptors, and levels of endogenous FGF were suppressed. Consistent with the presence of functional FGF receptors, addition of basic FGF overcame, at least in part, hypoxia‐mediated suppression of EC growth, and enhanced wound repair in hypoxia, stimulating both motility and cell division. Despite slower growth in hypoxia, ECs could achieve confluence, and the monolayers consisted of larger cells with altered assembly of the actin‐based cytoskeleton and small gaps between contiguous cells. The permeability of these hypoxic EC monolayers to macromolecules and lower molecular weight solutes was increased. Cell surface coagulant properties were also perturbed: the anticoagulant cofactor thrombomodulin was suppressed, and a novel Factor X activator appeared on the EC surface. These data indicate that micro‐ and macrovascular ECs can grow and be maintained at low oxygen tensions, but hypoxic endothelium exhibits a range of altered functional properties which can potentially contribute to the pathogenesis of vascular lesions.
Evidence for extralysosomal hydrolysis of high‐density lipoprotein cholesteryl esters in rat hepatoma cells (Fu5AH): A model for delivery of high‐density lipoprotein cholesterol
Authors: DeLamatre John G. , Carter Robert M. , Hornick Conrad A. 
Published: February 2005

AbstractRat hepatoma cells (Fu5AH) were studied as a model for the net delivery of apoE‐free high‐density lipoprotein (HDL) cholesterol to a cell. Incubating cells with HDL results in (1) a decrease in both media‐free cholesterol and cholesteryl ester concentration; (2) decreased cell sterol synthesis; and (3) increased cell cholesteryl ester synthesis. HDL cholesteryl ester uptake is increased when cells are incubated for 18 hr in cholesterol poor media. Coincubation of 3H‐cholesteryl ester‐labeled low‐density lipoprotein (LDL) with 50 μM chloroquine or 25 μM monensin results in a decrease in the cellular free cholesterol/cholesteryl ester (FC/CE) isotope ratio, indicating an inhibition in the conversion of cholesteryl ester to free cholesterol. In contrast, chloroquine and monensin do not alter the cellular FC/CE isotope ratio for 3H‐CE HDL. This evidence indicates that acidic lysosomal cholesteryl ester hydrolase does not account for the hydrolysis of HDL‐CE. Free cholesterol generated from 3H‐cholesteryl ester of both LDL and HDL is reesterified intracellularly. At higher HDL concentrations (above 50 μg/ml) HDL cholesteryl ester hydrolysis is sensitive to chloroquine. We propose that an extralysosomal pathway is operating in the metabolism of HDL cholesterol and that at higher HDL concentrations a lysosomal pathway may be functioning in addition to an extralysosomal pathway.
Characterization of atrial natriuretic peptide receptors in brain microvessel endothelial cells
Authors: Whitson Peggy A. , Huls M. Helen , Sams Clarence F. 
Published: February 2005

AbstractAtrial natriuretic peptide (ANP) binding and ANP‐induced increases in cyclic guanosine monophosphate (cGMP) levels have been observed in brain microvessels (Chabrier et al., 1987; Steardo and Nathanson, 1987), suggesting that this fluid‐regulating hormone may play a role in the fluid homeostasis of the brain. This study was initiated to characterize the ANP receptors in primary cultures of brain microvessel endothelial cells (BMECs). The apparent equilibrium dissociation constant, Kd, for ANP increased from 0.25 nM to 2.5 nM, and the number of ANP binding sites as determined by Scatchard analysis increased from 7,100 to 170,000 sites/cell between 2 and 10 days of culture following monolayer formation. Time‐ and concentration‐dependent studies on the stimulation of cGMP levels by ANP indicated that guanylate cyclase‐linked ANP receptors were present in BMECs. The relative abilities of ANP, brain natriuretic peptide (BNP), and a truncated analog of ANP containing amino acids 5–27 (ANP 5–27) to modulate the accumulation of cGMP was found to be ANP >BNP ≫ ANP 5–27. Affinity cross‐linking with disuccinimidyl suberate and radiolabeled ANP followed by gel electrophoresis under reducing conditions demonstrated a single band corresponding to the 60–70 kD receptor, indicating the presence of the nonguanylate cyclase‐linked ANP receptor. Radiolabeled ANP binding was examined in the presence of various concentrations of either ANP, BNP, or ANP 5–27 and suggested that a large proportion of the ANP receptors present in blood‐brain barrier endothelial cells bind all of these ligands similarly. These data indicate both guanylate cyclase linked and nonguanylate cyclase linked receptors are present on BMECs and that a higher proportion of the nonguanylate cyclase linked receptors is expressed. This in vitro culture system may provide a valuable tool for the examination of ANP receptor expression and function in blood‐brain barrier endothelial cells.
Extinction of the human insulin gene expression in insulinoma X fibroblast somatic cell hybrids involves cis‐acting DNA elements
Authors: Besnard Corinne , Monthioux Eliane , Loràs Patrick , Jami Jacques , Daegelen Dominique 
Published: February 2005

AbstractInsulin gene expression in rat insulinoma (RIN) cells is extinct in RIN × fibroblast hybrids and can reappear upon loss of DNA contributed by the fibroblast parent. (Besnard et al., Exp. Cell Res. 185:101–108, 1989). In the present study, we looked for the role of 5′‐flanking sequences of the human insulin gene in the negative control observed in the hybrids. RIN cells were transformed with composite genes which consisted of the coding sequence of the gpt gene placed under the control of 5′‐flanking regions of the human insulin gene (Ins. gpt gene). Upon hybridization of these cells with mouse fibroblasts, the expression of both Ins. gpt and endogenous rat insulin genes were suppressed together. The results obtained indicate that cis‐acting DNA elements are involved in the negative control of the gene. These elements are located in a fragment spread from ‐258 to +241 of the transcription origin of the human insulin gene.
Transforming growth factor‐β and platelet‐derived growth factor synergistically stimulate contraction by testicular peritubular cells in culture in serum‐free medium
Authors: Tung Pierre S. , Fritz Irving B. 
Published: February 2005

AbstractWe report investigations on factors influencing contractility by testicular peritu‐bular cells (PC) maintained in culture in a three‐dimensional collagen gel system, and the behavior of PC in culture on a two‐dimensional system. At low and moderate cell densities, PC embedded in collagen gels in serum‐free Eagle's minimal essential medium (MEM) have a lesser degree of contractility than PC in culture in MEM containing calf serum. The contractility by PC, measured by determining changes in diameter of the collagen gel, was increased by addition of transforming growth factor‐β (TGF‐β) to serum‐free MEM, and this was further enhanced by supplementing the medium with platelet‐derived growth factor (PDGF). In the absence of TGF‐β, however, PDGF had no detectable effects on PC contractility. Other growth factors examined (epidermal growth factor, insulin, and fibroblast growth factor) did not influence the degree of contractility of PC in serum‐free MEM in the presence or absence of TGF‐β. PC maintained in MEM supplemented with platelet‐poor serum (PPS) have a lesser degree of contractility than their counterparts in MEM containing 2.5% calf serum. The addition of TGF‐β and PDGF to PPS‐supplemented MEM restored contractility by PC to a level comparable to that observed by PC in MEM containing complete serum. The addition of nonpurified bovine serum albumin (BSA) to MEM greatly increased PC contractility. By contrast, highly purified BSA had no such effect, suggesting that one or more components adsorbed to the impure BSA was implicated. Polyclonal antibody against fibronectin did not influence the contractility of PC in collagen gels in the presence or absence of serum. Antiserum against TGF‐β partially blocked the enhancement of contractility of PC in MEM containing non‐purified BSA. In PC plated on top of a collagen gel lattice, the attachment, spreading, and cell shape were greatly influenced by the presence of TGF‐β and PDGF, both singly and together. Data presented are interpreted to indicate that effects elicited by serum on the properties of PC in culture, and on the contractility of PC, can be attributed in part to the combined influences of TGF‐β and PDGF in serum.
Serotonin: An inducer of collagenase in myometrial smooth muscle cells
Authors: Jeffrey John J. , Ehlich Lynn S. , Roswit William T. 
Published: February 2005

AbstractRat myometrial smooth muscle cells in culture actively produce collagenase in medium containing fetal bovine serum, but not in medium containing newborn bovine serum or containing fetal serum adsorbed with dextran‐coated charcoal. A dialyzable molecule has been isolated from fetal bovine serum, which restores the ability of the smooth muscle cells to produce collagenase. The molecule has been purified and identified as serotonin (5‐hydroxytryptamine). Cells cultured in medium depleted of serotonin for 3 days fail to produce collagenase, as assessed both enzymatically and immunologically. Addition of serotonin promptly restores the ability of the cells to produce the enzyme. The EC50 for serotonin is approximately 2 μM; maximum stimulation of collagenase production is observed at 5 μM. The response is specific for serotonin: a wide variety of compounds tested, either related to serotonin or of potential eproductive significance, were without effect in the induction of collagenase production by the cells. No changes in DNA content, general protein synthesis, or cellular collagen production were observed as a consequence of serotonin depletion or restoration, suggesting a selective effect of the compound on collagenase production. The effect of serotonin was also selective to myometrial smooth muscle cells; collagenase‐producing fibroblasts from skin and cervix displayed no serotonin requirement for enzyme production. Studies using specific agonists or antagonists for a variety of serotonin receptor subtypes suggest that the 5‐HT‐2 receptor mediates the serotonin induction of collagenase in these cells. Preliminary evidence indicates that cultured human myometrial smooth muscle cells are also dependent upon serotonin for collagenase production. The evidence in this study suggests the possibility that serotonin serves as a signal to begin the massive collagen degradation that occurs in the postpartum uterus.
Chloride secretagogues stimulate inositol phosphate formation in shark rectal gland tubules cultured in suspension
Authors: Ecay Tom W. , Valentich John D. 
Published: February 2005

AbstractNeuroendocrine activation of transepithalial chloride secretion by shark rectal gland cells is associated with increases in cellular cAMP, cGMP, and free calcium concentrations. We report here on the effects of several chloride secretagogues on inositol phosphate formation in cultured rectal gland tubules. Vasoactive intestinal peptide (VIP), atriopeptin (AP), and ionomycin increase the total inositol phosphate levels of cultured tubules, as measured by ion exchange chromatography. Forskolin, a potent chloride secretagogue, has no effect on inositol phosphate formation. The uptake of 3H‐myo‐inositol into phospholipids is very slow, preventing the detection of increased levels of inositol trisphosphate. However, significant increases in inositol monophosphate (IP1) and inositol biphosphate (IP2) were measured. The time course of VIP‐ and AP‐stimulated IP1 and IP2 formation is similar to the effects of these agents on the short‐circuit current responses of rectal gland monolayer cultures. In addition, aluminum fluoride, an artificial activator of guanine nucleotide‐binding proteins, stimulates IP1 and IP2 formation. We conclude that rectal gland cells contain VIP and AP receptors coupled to the activation of phospholipase C. Coupling may be mediated by G‐proteins. Receptor‐stimulated increases in inositol phospholipid metabolism is one mechanism leading to increased intracellular free calcium concentrations, an important regulatory event in the activation of transepithelial chloride secretion by shark rectal gland epithelial cells.
Exosome formation during maturation of mammalian and avian reticulocytes: Evidence that exosome release is a major route for externalization of obsolete membrane proteins
Authors: Johnstone R. M. , Mathew A. , Mason A. B. , Teng K. 
Published: February 2005

AbstractWe have assessed whether exosome formation is a significant route for loss of plasma membrane functions during sheep reticulocyte maturation in vitro.Although the recovery of transferrin binding activity in exosomes is at best ∼25–30% of the lost activity, recoveries of over 50% of the lost receptor can be obtained if 125I‐labelled transferrin receptor is measured using an immunological approach. Degradation products of the transferrin receptor in the medium suggest that receptor instability may contribute to the less than quantitative recovery of the transferrin receptor. Significantly higher (75–80%) levels of the nucleoside transporter can be recovered in exosomes during red cell maturation using 3H‐nitrobenzylthioinosine binding to measure the nucleoside transporter.These data suggest that exosome formation is a major route for removal of plasma membrane proteins during reticulocyte maturation and plasma membrane remodelling.We have also shown that both in vivo and in vitro, embryonic chicken reticulocytes form exosomes which contain the transferrin receptor. Thus, exosome formation is not restricted to mammalian red cells, but also occurs in red cells, which retain organelles, such as nuclei and mitochondria, into the mature red cell stage.
Insulin‐induced mitogenesis associated with transformation by the SV40 large T antigen
Authors: Wang Hanlin , Scott Robert E. 
Published: February 2005

AbstractSimian virus 40 (SV40) large T antigen‐transformed cells typically show a markedly reduced serum requirement for growth and the inability to growth arrest and differentiate. An SV40 large T antigen‐transformed 3T3 T cell line, CSV3‐1, that can growth arrest and differentiate into adipocytes with high efficiency has, however, recently been described (Scott et al: Proc. Natl. Acad. Sci. U.S.A. 86: 1652–1656, 1989; Estervig et al: J. Virol. 63: 2718–2725, 1989; J. Cell. Physiol. 142:552–558, 1990). The results of the current studies using these cells show that whereas quiescent 3T3 T cells show no mitogenic response to insulin, quiescent CSV3‐1 cells show a highly significant insulin‐induced mitogenic responsiveness in the absence of other added growth factors. Maximum mito‐genesis was observed at an insulin concentration of 1 μg/ml, which induced 40–70% of the cells to undergo DNA synthesis within 48 hours. The half maximum response was achieved with 1–10 ng/ml of insulin. Insulin's mitogenic effect on CSV3‐1 cells was evident under several different culture conditions that induce quiescence and was not mediated by any detectable autocrine growth factors that might make CSV3‐1 cells competent to respond to insulin. In CSV3‐1 cells insulin appears to act on its own receptor rather than on the IGF‐1 receptor, because at comparable dosages IGF‐1 is 10‐to 100‐fold less effective than insulin. Insulin also is shown to be a mitogen for another SV40‐transformed cell line, CSV3–35, which can be growth arrested; in contrast insulin has no mitogenic effect on two control cell lines that are stably transfected with pSV2neo, a plasmid containing SV40 early promoter/enhancer but lacking large T antigen gene. These results suggest a significant relationship between SV40 T antigen‐associated transformation and the expression of mitogenic responsiveness to insulin.
Regulatory mechanisms for thrombomodulin expression in human umbilical vein endothelial cells in vitro
Authors: Hirokawa Kazunori , Aoki Nobuo 
Published: February 2005

AbstractIt has been reported that thrombomodulin (TM) expression in endothelial cells is modulated by various agents. We investigated cellular regulatory mechanisms for TM expression in human umbilical vein endothelial cells (HUVECs), incubated with agents, by measuring the time course changes in surface TM activity, total TM antigen in cell lysates, and TM mRNA levels. While dibutyryl cAMP (3 mM) increased TM mRNA levels in HUVECs and was followed by increased TM activity, dibutyryl cGMP had no effect on TM activity. Phorbol myristate acetate (PMA) induced rapid loss of surface TM activity (∼8 h) and later increased TM mRNA levels between 4 h and 40 h (maximum at 24 h), resulting in biphasic effects on TM activity. Tumor necrosis factor or interleukin‐1β suppressed surface TM activity and TM mRNA levels. Internalization/degradation of TM in HUVECs incubated with PMA or cytokines was suggested by co‐culture with chloroquine. The decrease in surface TM activity observed was not caused by the release of TM molecules from the cells into the conditioned media. These results suggest that TM activity in HUVECs is modulated by independent mechanisms involving cytoplasmic TM mRNA levels and internalization/degradation of TM molecules. These regulatory mechanisms may involve protein kinase A and protein kinase C‐dependent mechanisms but are independent of protein kinase G.