bluuebunny/crossref_metadata_2025_split huggingface dataset:
Secretion of a TGF‐β‐like growth inhibitor by normal rat mammary epithelial cells in vitro
Authors: Ethier Stephen P. , Van De Velde Rochelle M. 
Published: February 2005

AbstractWe have examined conditioned medium (CM) from cultures of normal rat mammary epithelial (RME) cells for growth factor activity on fresh RME cell cultures. RME cell‐derived CM contained potent growth inhibitory activity toward fresh RME cell cultures when the medium was acidified by dialysis against 1% acetic acid prior to concentration. Dialysis of the CM at neutral pH resulted in CM that had growth stimulatory activity and no inhibitory activity. The acid‐activated growth inhibitor was heat and acid stable, protease sensitive, and eluted from a Bio‐Gel p60 column with a peak of activity in the 28 kDa range. Incubation of the acidified‐concentrated CM with neutralizing antiserum (affinity purified IgG) against transforming growth factor (TGF)‐β completely abolished the inhibitory activity of the CM. Furthermore, RME cell growth in the presence of the growth inhibitor plus TGF‐β antiserum was greater than that observed in growth medium alone. Subsequent experiments demonstrated that addition of TGF‐β antiserum alone to serum‐free medium enhanced RME cell growth, whereas addition of nonimmune IgG was without effect even at 25‐fold higher concentrations. Zymographic analysis of RME‐CM revealed the presence of plasminogen activator proteases that may mediate the partial activation of the latent growth factor. These results indicate that normal RME cells secrete a latent TGF‐β‐like growth factor into conditioned medium. Furthermore, the results indicate that some of the latent growth factor is activated in situ and contributes to the growth potential of the cells in primary culture in an autocrine manner.
Release of calcium from intracellular stores in rat basophilic leukemia cells monitored with the fluorescent probe chlortetracycline
Authors: Marcotte Gregory V. , Millard Paul J. , Fewtrell Clare 
Published: February 2005

AbstractRelease of calcium from intracellular stores of rat basophilic leukemia cells was monitored using the fluorescent probe chlortetracycline. The ability of chlortet‐racycline to indicate release from intracellular calcium stores was initially validated. The decrease of chlortetracycline fluorescence upon antigen‐stimulation was not the result of secretion of granule‐associated dye or of changes in the properties of the membranes. The chlortetracycline fluorescence signal was not influenced by Ca2+ influx across the plasma membrane. Results obtained from these chlortetracycline fluorescence measurements corresponded well with 45Ca efflux data, an indirect measurement of release of calcium from stores. Chlortet‐racycline was used to examine the rate of antigen‐induced release of calcium from stores, the depletion of intracellular calcium stores by EGTA, and the relationship between the antigen‐stimulated release of stored calcium and exocytosis. Chlortetracycline was shown to be a useful qualitative indicator for the release of intracellular calcium with a relatively rapid response time.
Transforming growth factor beta1 modulates extracellular matrix organization and cell‐cell junctional complex formation during in vitro angiogenesis
Authors: Merwin June Rae , Anderson James M. , Kocher Olivier , Van Itallie Christina M. , Madri Joseph A. 
Published: February 2005

AbstractTransforming growth factor‐beta1 (TGF‐β1) is angiogenic in vivo. In two‐dimensional (2‐D) culture systems microvascular endothelial cell proliferation is inhibited up to 80% by TGF‐β1 however, in three‐dimensional (3‐D) collagen gels TGF‐β1 is found to have no effect on proliferation while eliciting the formation of calcium and magnesium dependent tube‐like structures mimicking angiogenesis. DNA analyses performed on 3‐D cell cultures reveal no significant difference in the amount of DNA or cell number in control versus TGF‐β1 treated cultures. In 2‐D cultures TGF‐β1 is known to increase cellular fibronectin accumulation; however, in 3‐D cultures no difference is seen between control and TGF‐β1 treated cells as established by ELISA testing for type IV collagen, fibronectin, and laminin. In 3‐D cultures there is increased synthesis and secretion of type V collagen in both control and TGF‐β1 treated cultures over 2‐D cultures. Even though an equal amount of type V collagen is seen in both 3‐D conditions, there is a reorganization of the protein with concentration along an organizing basal lamina in TGF‐β1 treated cultures. EM morphological analyses on 3‐D cultures illustrate quiescent, control cells lacking cell contacts. In contrast, TGF‐β1 treated cells show increased pseudopod formation, cell‐cell contact, and organized basal lamina‐like material closely apposed to the “abluminal” plasma membranes. TGF‐β1 treated cells also appear to form junctional complexes between adjoining cells. Immunofluorescence using specific antibodies to the tight junction protein ZO‐1 results in staining at apparent cell‐cell junctions in the 3‐D cultures. Northern blots of freshly isolated microvascular endothelium, 2‐D and 3‐D cultures, using cDNA and cRNA probes specific for the ZO‐1 tight junction protein, reveal the presence of the 7.8 kb mRNA. Western blots of rat epididymal fat pad endo‐thelial cells (RFC) monolayer lysates probed with anti‐ZO‐1 label a 220 kd band which co‐migrates with the bonafide ZO‐1 protein. These data confirm and support the hypothesis that TGF‐β1 is angiogenic in vitro, eliciting microvascular endothelial cells to form tube‐like structures with apparent tight junctions and abluminal basal lamina deposition in three‐dimensional cultures.
Augmented desensitization to epidermal growth factor (EGF) immediate actions: A novel mechanism for altered EGF growth response in mutant A431 cells
Authors: Karasik Avraham , Reddy Sethu S.‐K. , Pepinsky R. Blake , Brock Tommy , Kahn C. Ronald 
Published: February 2005

AbstractEpidermal growth factor (EGF) may either stimulate or inhibit cell growth. To elucidate the mechanism of these varied effects, we compared EGF action in parental A431 cells in which cell growth is inhibited, and clone 15, a mutant of these cells resistant to EGF growth inhibition. In both lines, EGF receptor was present in similar concentrations and underwent tyrosine phosphorylation to the same extent. Likewise, in both lines, acute exposure to EGF stimulated an increase in free cytoplasmic [Ca2+], as well as a similar increase in phosphoryla‐tion of lipocortin 1, a major substrate for the EGF receptor kinase whose phos‐phorylation is calcium‐dependent. On the other hand, pretreatment of clone 15 cells with EGF for 72 h abolished EGF‐induced phosphorylation of lipocortin 1 and led to a loss of the increase in cytoplasmic free [Ca2+], whereas no such desensitization was seen in the parental A431 cells. These data indicate a link between EGF‐induced increase in cytoplasmic calcium, lipocortin phosphoryla‐tion, and cell growth and suggest that differences in mechanisms of desensitization to these immediate actions of EGF may lead to altered growth response to this hormone.
Fusion of mouse alveolar macrophages induced by 1α,25‐dihydroxyvitamin D3 involves extracellular, but not intracellular, calcium
Authors: Jin Cheng He , Miyaura Chisato , Tanaka Hirofumi , Takito Jiro , Abe Etsuko , Suda Tatsuo 
Published: February 2005

AbstractWe have reported that the active form of vitamin D3, 1α, 25‐dihydroxy‐vitamin D3 [1α,25(OH)2D3], directly induces the fusion of mouse alveolar macrophages (Abe et al: Proc. Natl. Acad. Sci. USA 80:5583–5587, 1983). The fusion process can be divided into two phases: the 1α,25(OH)2D3‐dependent priming phase (0–18 hr) and the calcium‐dependent progression phase (18–72 hr) (Jin et al: Cell. Physiol.137:110–116, 1988). In the present study, we examined the role of calcium in the progression phase of macrophage fusion induced by 1α,25(OH)2D3. Macrophages pretreated with 1α,25(OH)2D3 for 48 hr in a lowcalcium (0.13 mM) medium began to fuse quickly 30 min after the culture medium was switched to a normal calcium (1.85 mM) medium. Of various cations tested, calcium was the most effective in inducing fusion, followed by strontium and manganese. Magnesium, potassium, and sodium had no effect. Calcium ionophores such as A23187 and ionomycin did not induce fusion in the lowcalcium medium, nor did they potentiate fusion in the media containing higher concentrations of calcium. The intracellular concentration of free Ca2+, measured by a fluorescent method using fura‐2 AM, was 116 ± 1 nM in the macrophages pretreated with 1α,25(OH)2D3 for 48 hr in the low‐calcium medium. When calcium chloride was added to the assay system at a final concentration of 1.85 mM, the cytosolic free Ca2+ concentration did not increase appreciably (from 116 to 144 nM). But the macrophages began to fuse quickly when CaCI2 was added. In contrast, adding ionomycin increased cytosolic free Ca2+ from 116 to 440 nM, but no fusion occurred. These results clearly indicate that the extracellular, but not the intracellular, calcium is involved in the progression phase of the fusion of mouse alveolar macrophages primed by 1α,25(OH)2D3.
Phorbol ester regulation of terminal deoxynucleotidyl transferase, proliferation, and TcR α in a pre‐T cell line
Authors: Beland Janice L. , Martin A. R. Yuille , Hugunin Margaret , Zhang Xinming , Silverstone Allen E. 
Published: February 2005

AbstractTerminal deoxynucleotidyl transferase (TdT) is a template‐independent DNA polymerase that is transiently expressed during the normal development of T and B lymphocytes. Phorbol 12‐myristate 13‐acetate (PMA) has been reported to induce maturation‐like changes, including the loss of TdT, in many leukemic cell lines. We investigated the mechanism of TdT repression by PMA in an early thymocyte‐like cell line, RPMI 8402. At a concentration of 8 nM, PMA caused both repression of TdT synthesis and arrest of proliferation. At greater concentrations of PMA, these same changes initially occurred, but then cell proliferation resumed, and TdT was reexpressed. At both 8 and 160 nM PMA, TdT biosynthesis and TdT mRNA became undetectable within 8 hours, while cell proliferation and DNA synthesis were not significantly reduced until 16 hours. Growth arrest induced by serum starvation did not result in a similar reduction of TdT RNA even after 48 hours. With 160 nM PMA, TdT mRNA could be detected again by 24 hours, and proliferation resumed. Transcription run‐off assays indicated that TdT RNA synthesis ceased within 1 hour after exposure to both 8 and 160 nM PMA. T cell receptor α (TcR α) RNA was induced when TdT RNA was repressed. TcR β RNA levels were unchanged, and TcR γ RNA was up‐regulated. TdT gene repression and modulation of cell proliferation as well as induction of TcR gene expression are normal events during intrathymic T cell maturation. This cell model provides a system for analyzing the molecular regulation of these significant developmental events.
Interrelationship between signals transduced by phytohemagglutinin and interleukin 1
Authors: Mills Gordon B. , Hill Mary , McGill Martha , May Christopher , Stanley Jacqueline , Stewart David J. , Mellors Alan , Gelfand Erwin W. 
Published: February 2005

AbstractIn the murine cell line LBRM‐331A5, phytohemagglutinin (PHA) induces secretion of the T cell growth factor interleukin 2 (IL2). IL1 augments PHA‐induced IL2 production. In this cell line, PHA stimulates a number of biochemical changes including phospholipid hydrolysis, increases in cytosolic free calcium ([Ca2+]i), membrane hyperpolarization, cytosolic alkalinization, and tyrosine phosphorylation of specific substrates. Using LBRM cells, we have studied the interrelationship between these events and the secretion of IL2. Increases in [Ca2+]i triggered by PHA or following addition of ionomycin result in membrane hyperpolarization but are not required for PHA‐induced cytosolic alkalinization or tyrosine phosphorylation. Addition of IL1 to PHA‐stimulated cells did not affect any of the biochemical parameters, although it significantly augmented PHA‐induced IL2 secretion. Increasing [Ca2+]i with ionomycin did not trigger IL2 secretion, increases in cytosolic pH, or tyrosine phosphorylation in the presence or absence of IL1. Preventing increases in cytosolic pH did not alter PHA‐induced changes [Ca2+]i in or membrane potential. These data are compatible with PHA including activation of phospholipase C and production of inositol phosphates resulting in both release of Ca2+ from internal stores and transmembrane uptake of Ca2+ as well as activation of protein kinase C. However, unlike other growth factor or mitogen‐stimulated systems, the changes stimulated by PHA and IL1 in LBRM cells including IL2 secretion are not regulated by a pertussis toxin‐sensitive G protein.
Evidence for poliovirus‐induced cytoplasmic alkalinization in Hela cells
Authors: Holsey Charles , Edward J. Cragoe , Nair C. N. 
Published: February 2005

AbstractDuring the early period after poliovirus infection of HeLa cells, cellular Na+/K+ ATPase activity is transiently activated. We investigated the possibility that Na+/K+ ATPase activation is a consequence of Na+/H+ antiporter activation. Increased uptake of the weak organic acid 5,5‐dimethyloxazolidine‐2,4‐dione by infected cells around 2 h after infection suggested cytoplasmic alkalinization equivalent to pH 7.7 during the biosynthetic phase of viral replication. Consistent with the involvement of Na+/H+ antiporter activation in this phenomenon, it was found to be [Na+]‐dependent and inhibited by 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA). However, the pH increase was not associated with an increase in amiloride‐sensitive Na+ uptake by infected cells predicted by this mechanism. By contrast, the alkalinization could be abolished with the anion‐exchange inhibitor 4,4′‐diisothiocyanostilbene‐2,2'‐disulfonic acid (DIDS), implicating an anion‐exchange mechanism, such as CI−/HCO3− exchange, in this process. In addition to abolishing virus‐induced intracellular alkalinization, both EIPA and DIDS moderately inhibited viral replication. Manipulation of intracellular pH with nigericin in the incubation medium revealed that maximum viral replication required a pH of about 7.7 and that replication was significantly inhibited even at pH 7.3. Thus, the pH increase in infected cells appeared to be physiologically relevant. These findings represent the first demonstration of a biologically meaningful pH increase in cells infected with a lytic virus.
Correlation between contractility and proliferation in human fibroblasts
Authors: Macieira‐Coelho A. , Azzarone B. 
Published: February 2005

AbstractThe contractile power of human fibroblasts was checked through their life span in vitro, using a plasma clot retraction test. It was found to decline with a pattern analogous to that of the different phases identifiable by the study of the kinetics of proliferation of these cells. The capacity to retract a plasma clot was higher in cells harvested during active growth than in cells harvested in resting phase. The decreased ability to retract during aging becomes apparent when cells are harvested in resting phase. Decreased retractile activity was also observed in postnatal cells as compared with embryonic cells. The results support a correlation between the initiation of DNA synthesis and the turnover of cytoskeletal elements. The data fit our previous results showing that the early proliferative disturbance during cellular senescence consists of a decline in the probability of initiating the division cycle linked to impaired cell attachment and spreading.
Increased thermostability of thermotolerant CHL V79 cells as determined by differential scanning calorimetry
Authors: Lepock James R. , Frey Harold E. , Heynen Miriam P. , Nishio John , Waters Brenda , Ritchie Kenneth P. , Kruuv Jack 
Published: February 2005

AbstractHeat shock denatures cellular protein and induces both a state of acquired thermotolerance, defined as resistance to a subsequent heat shock, and the synthesis of a category of proteins referred to as heat‐shock proteins (HSPs). Thermotolerance may be due to the stabilization of thermolabile proteins that would ordinarily denature during heat shock, either by HSPs or some other factors. We show by differential scanning calorimetry (DSC) that mild heat shock irreversibly denatures a small fraction of Chinese hamster lung V79‐WNRE cell protein (i. e., the enthalpy change, which is proportional to denaturation, on scanning to 45°C at 1°C/min is approximately 2.3% of the total calorimetric enthalpy). Thermostability, defined by the extent of denaturation during heat shock and determined from DSC scans of whole cells, increases as the V79 cells become thermotolerant. Cellular stabilization appears to be due to an increase in the denaturation temperature of the most thermolabile proteins; there is no increase in the denaturation temperatures of the most thermally resistant proteins, i. e., those denaturing above 65°C. Cellular stabilization is also observed in the presence of glycerol, which is known to increase resistance to heat shock and to stabilize proteins in vitro. A model is presented, based on a direct relationship between the extent of hyperthermic killing and the denaturation or inactivation of a critical target that defines the rate‐limiting step in killing, which predicts a transition temperature (Tm) of the critical target for control V79‐WNRE cells of 46.0°C and a Tm of 47.3°C for thermotolerant cells. This shift of 1.3°C is consistent with the degree of stabilization detected by DSC.
Platelet‐derived growth factor regulates actin isoform expression and growth factor regulates actin isoform expression and growth state in cultured rat aortic smooth muscle cells
Authors: Blank Randal S. , Owens Gary K. 
Published: February 2005

AbstractThe role of platelet‐derived growth factor (PDGF) in the control of smooth muscle cell (SMC) differentiation was explored in vitro by examining its effects on expression of the smooth muscle (SM) specific contractile protein SM alpha actin in cultured rat aortic SMC. Quiescent, postconfluent SMC express maximal levels of alpha actin and responded to human platelet‐derived growth factor (partially purified from platelets) by entering the cell cycle and undergoing approximately one synchronous round of DNA synthesis. Concomitantly these cultures exhibited a marked reduction in alpha actin synthesis. Chronic treatment with PDGF (72 hours at 8 or 12 hour intervals) was associated with a transient increase in thymidine labeling index and a decrease in alpha actin expression. Interestingly, between 48 and 72 hours following initial treatment, thymidine labeling indices returned to near control levels while SM alpha actin expression remained depressed. This effect was reversible; fractional alpha actin synthesis increased immediately after PDGF removal. When subsequently stimulated with 10% fetal bovine serum (FBS), cells chronically pretreated with PDGF entered S phase approximately 4 hours earlier than cells pretreated with PDGF vehicle, consistent with the idea that the maintained suppression of alpha actin synthesis in SMC subjected to chronic PDGF treatment was associated with partial cell cycle transit. Chronic treatment with highly purified recombinant PDGF‐BB elicited similar effects on alpha actin synthesis and partial cell cycle transit. Flow cytometric analysis of chronic PDGF‐treated SMC demonstrated a 25% increase in forward angle light scatter, an index of cell size. These data implicate a possible role for PDGF in regulation of SMC differentiation and suggest a potentially important role for this mitogen in the phenotypic modulation accompanying SMC growth and in mediation of the cellular hypertrophy associated with cell cycle progression.
Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network
Authors: Raub Thomas J. , Koroly Mary Jo , Roberts R. Michael 
Published: February 2005

AbstractBy using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi‐thin sections by medium‐ and high‐voltage electron microscopy revealed the three‐dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate‐, horseradish peroxidase‐, or ferritin‐conjugated WGA to cells at 4°C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface‐labeled cells to 37°C resulted in the endocytosis of WGA into peripheral endo‐somes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300‐400 nm diam.) surrounded by and continuous with tubular cister‐nae (45‐60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45‐60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA‐HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans‐Golgi region. The accumulation of WGA‐HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18°C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans‐Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post‐endosomal, tubular reticulum that appears to be separate from the trans‐most Golgi saccule.