bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification, characterization, and localization of 70 kDa calcium‐sensitive protein (calelectrin) from mammary glands
Authors: Lozano Joseph J. , Haindl Alfred H. , Rocha Victor
Published: February 2005

Mammary glands contain a group of calcium‐sensitive proteins that bind to membranes in a calcium‐dependent manner. Using the calcium‐dependent binding to hydrophobic surfaces in combination with conventional techniques, we have purified the 70kDa mammary calcium‐binding protein (70 kDa M‐CBP) to homogeneity. Antisera prepared to the 70 kDa M‐CBP or to bovine liver 67 kDa calelectrin reacted in immunoblot analysis with the 70 kDa M‐CBP antigen and with several additional mammary CBP species in crude tissue homogenates. Limited proteolysis of the 70 kDa M‐CBP produced smaller immunoreactive species; extensive proteolysis resulted in more complete degradation of the protein. Identical data were obtained with digestion of 67 kDa calelectrin. The pi for the 70 kDa M‐CBP was determined to be approximately 5.8; the same value reported for 67 kDa calelectrin. Phosphorylation of 70 kDa M‐CBP was not detected in epithelial cell culture metabolic labeling. Immunohistochemical localization showed the protein to be located in ductal epithelia of virgin mouse mammary glands with a pattern of increased staining of the basal portions of the cells. Some stromal cells were also reactive. Apparently, the 70 kDa M‐CBP and 67 kDa calelectrin are the same protein. Furthermore, like the 32.5 calelectrin (endonexin) and calpactin 1/p36/lipocortin II, the 70 kDa protein appears to be a ductal epithelial cell associated protein in the mammary gland.
Structure of transcriptionally active chromatin: Radiological evidence for requirement of torsionally constrained DNA
Authors: Rodi Charles P. , Sauerbier Walter
Published: February 2005

Synthesis of α‐ and β‐globin RNA in DMSO‐induced Friend's erythroleukemia cells and synthesis of immunoglobulin γ‐ and k‐chain RNA, total RNA, 5S RNA, and tRNA in mouse myeloma cells (MPC‐11) was inhibited by γ‐irradiation. For all RNA species, synthesis decreased nearly exponentially as a function of radiation dose, whereas RNA size distributions, turnover rates, and specific activities of radioactively labeled RNA were affected only insignificantly. D37 values for the loss of synthesis of various RNA species correspond to target sizes ranging from 21,000 to 53,000 kd, or 30–80 kbp of DNA. These target sizes are several‐fold larger than the structural genes in question; however, they correspond well with the size of DNA loops, or “domains” constrained by the nuclear matrix. The data suggest that the eukaryotic transcription unit is the torsionally constrained chromatin loop, transcription of which may be inactivated, or significantly reduced by a DNA single‐strand break.
Clonal interactions in fibroblast proliferation: Recognition of self vs. non‐self
Authors: Korn Joseph H. , Downie Elaine
Published: February 2005

The proliferation and aging of fibroblast populations has been postulated to include a process of clonal selection. Using carbocyanine dyes to label clonal fibroblast populations, we were able to follow their growth in mixed cultures. Individual fibroblast clones seeded as the minority population (20%) with either another clone or the parent line (differentially labeled) always demonstrated increase relative growth so that, by the end of 4 weeks, approximately equal numbers of both populations were present. Labeled clones mixed as the minority population with differentially labeled cells of the same clone maintained their minority status. The results indicate that clonal populations of fibroblasts are able to recognize “self” as different from “non‐self” and that this recognition leads to alterations in cellular proliferation.
Interferon α/β modulation of growth‐factor‐stimulated mitogenicity in AKR‐2B fibroblasts
Authors: Pietenpol Jennifer A. , Howe Philip H. , Cunningham Muriel R. , Leof Edward B.
Published: February 2005

Growth factor‐stimulated mitogenicity in mouse embryo‐derived AKR‐2B cells was inhibited in a dose‐dependent fashion by a mixture of alpha and beta mouse interferons (IFN). A 60% decrease in epidermal growth factor (EGF) and insulin‐stimulated DNA synthesis was observed with 10 kU/ml IFN, and half‐maximal inhibition was seen at 1 kU/ml. Likewise, the mitogenic effect of 5% fetal bovine serum (FBS) was inhibited by 60% with 10 kU/ml IFN and by 38% with 1 kU/ml IFN. IFN inhibition of DNA synthesis was paralleled by a decrease in monolayer growth of AKR‐2B cells by 60% on the 3rd day of culture and by 40% on the 6th day of culture. Soft agar growth of two AKR‐2B derived lines, AKR‐MCA and AKR‐2B (clone 84A), was also inhibited significantly with the addition of 1–10 kU/ml of IFN. The effect of IFN on EGF receptors was also examined. Treatment of AKR‐2B cells with 10 kU/ml IFN resulted in a 35% decrease in EGF binding to cell surface receptors. The reduced binding of EGF to cells treated with IFN was due to a loss of EGF receptors as determined by Scatchard analysis. IFN treatment of AKR‐2B cells neither altered the affinity of the EGF receptor for its ligand nor affected receptor internalization. Nuclear transcription and actinomycin D decay analysis indicated that within 2 hr, IFN reduced c‐myc messenger RNA levels at the level of transcription with no affect on message decay.
Cell surface distribution of Fc receptors II and III on living human neutrophils before and during antibody dependent cellular cytotoxicity
Authors: Petty Howard R. , Francis Joseph W. , Anderson Clark L.
Published: February 2005

Microscopic techniques have been employed to study the cell surface distributions of the immunoglobulin Fc receptors (FcR) II and III on living human neutrophils. Fluorescein‐ or rhodamine‐conjugated monoclonal IgG or Fab fragments directed against FcRII (CDw32) and FcRIII (CD16) were employed to label receptors. FcRII and III were found to be uniformly distributed at neutrophil surfaces during resting conditions. During neutrophil polarization and migration FcRII but not FcRIII preferentially accumulated at the uropod. Sheep erythrocytes (SRBCs) were opsonized with IgG and then incubated with neutrophils. When neutrophils were labeled prior to target addition, FcRII but not FcRIII were found to cluster at the target‐effector interface. Little or no clustering of FcRs was observed if labeling was performed after target binding. SRBC oxidation was observed using Soret band illumination during transmitted light microscopy. Time‐lapse studies of FcRII distribution and target oxidation were performed. FcRII formed clusters at target‐effector interfaces prior to target oxidation. Three lines of evidence suggest that clustering is not a general plasma membrane response. Firstly, FcRIII do not cluster. Tannic acid‐modified erythrocytes avidly bound to neutrophils but did not trigger clustering of FcRII. Furthermore, irrelevant neutrophil membrane labels were unaffected by the presence of IgG‐opsonized erythrocytes. We suggest that FcRII clustering is one important component leading to the oxidative destruction of target cells.
Preparation and characterization of plasma membrane vesicles from human polymorphonuclear leukocytes
Authors: Del Buono Brian J. , Luscinskas Francis W. , Simons Elizabeth R.
Published: February 2005

It would be advantageous to prepare models of the neutrophil plasma membrane in order to examine the role of the plasma membrane in transmembrane signal transduction in the human neutrophil and to dissect ligand‐receptor interactions and structural changes in the cell surface upon stimulation. A number of investigators have prepared neutrophil membrane vesicles by homogenization, sonication, or centrifugation–techniques that can result in the loss of substantial amounts of surface membrane material, disruption of lysosomes causing proteolysis of membrane proteins, and contamination of the plasma membrane fraction by internal membranes. These limitations have been overcome in the present studies by employing a modification of the method previously developed in this laboratory. Human neutrophils were suspended in a buffer simulating cytoplasmic ionic and osmotic conditions and disrupted by nitrogen cavitation. The resultant cavitate was freed of undisrupted cells and nuclei and then centrifuged through discontinuous isotonic/isoosmotic Percoll gradients, which resolved four fractions: α (intact azurophilic granules), β (intact specific granules), γ (membrane vesicles), and δ (cytosol). The γ fraction was highly enriched in alkaline phosphatase, a marker of the plasma membrane. In addition, this fraction contained <5% of the amounts of lysosomes (indicated by lysozyme activity) and nuclei (indicated by DNA content) found in intact cells or in unfractionated cavitate. Furthermore, the γ fraction contained <10% of the levels of endoplasmic reticulum, Golgi, mitochondrial, and lysosomal membranes in cells or cavitates, as determined by assays for glucose 6‐phosphatase, galactosyl transferase, monoamine oxidase, and Mo1 (CD11b/CD18; Mac‐1), respectively. Finally, 75% of the membrane vesicles were sealed, as indicated by assay of ouabain‐sensitive (Na+, K+) ATPase activity, and 55% were oriented right‐side‐out, as determined by exposure of concanavalin A (ConA) receptors and sialic acid residues on the surfaces of the vesicles. These heterogeneous preparations could be enriched for right‐side‐out vesicles by their selective adherence to ConA‐coated plates and subsequent detachment by rinsing the surfaces of the plates with α‐methylman‐noside. This enrichment protocol did not affect the integrity of the vesicles and resulted in populations in which >85% of the vesicles were oriented right‐sideout. This procedure thus permits the preparation of sealed, right‐side‐out membrane vesicles that may be used as valid experimental models of the neutrophil plasma membrane in a variety of functional studies.
Synthesis and secretion of interphotoreceptor retinoid‐binding protein (IRBP) by isolated normal and rd mouse retinal photoreceptor neurons in culture
Authors: Politi L. E. , Lee L. , Wiggert B. , Chader G. , Adler R.
Published: February 2005

Cultures of dissociated retinal neurons and photoreceptors from homozygous wild‐type, heterozygous rd/+ and homozygous rd/rd retinas have been used to investigate the capacity of isolated photoreceptor cells to synthesize and secrete the interphotoreceptor retinoid‐binding protein (IRBP). Retinal cells were dissociated on postnatal day 2 and grown in chemically defined medium in the absence of glial and pigmented epithelial cells. Expression of IRBP immunoreactive materials in these cultures was cell type‐specific and developmentally regulated. Thus increasing numbers of rod photoreceptor cells showed immunoreactivity during the first week in culture, whereas nonphotoreceptor cell types remained consistently negative. Photoreceptor immunoreactivity could be detected in permeated (detergent‐treated) as well as in unpermeated preparations, the latter suggesting that some IRBP is associated with the photoreceptor cell surface. These materials appeared to be loosely bound to the photoreceptors, since they disappeared when the cultures were exposed for 6 hr to IRBP‐free medium but not when they were exposed to IRBP‐containing medium. IRBP synthesis and secretion could be demonstrated by analyzing either cell extracts or conditioned medium by “slot blot” and Western blot techniques using affinity purified antibodies against bovine IRBP as well as by fluorographic analysis after metabolic labeling of the cultures with 35S‐methionine. Comparisons of cultures from the different genotypes showed many similarities, including the abundance of IRBP‐immunoreactive photoreceptors in 7 day cultures. However, immunochemical analysis showed lower conditioned medium/cell extract IRBP ratios in rd/rd cultures, an observation consistent with previous reports suggesting that IRBP secretion may be deficient in rd/rd photoreceptor cells.
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

We 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

Release 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

Transforming 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

Epidermal 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

We 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

Terminal 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

In 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

During 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

The 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

Heat 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

The 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

By 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.
Potentiation of oncogenic N‐ras‐induced neurite outgrowth and ornithine decarboxylase activity by phorbol dibutyrate and protein kinase inhibitor H‐8
Authors: Trotta Robert J. , Thomson Timothy M. , Lacal Juan‐Carlos , Pellicer Angel , Burstein David E.
Published: February 2005

A recombinant N‐ras oncogene, under the transcriptional control of a corticosteroid‐inducible mouse mammary tumor virus (MMTV) promoter, has been stably transfected into a PC12 rat pheochromocytoma subline. This cell line, designated UR61, undergoes N‐ras‐induced neurite outgrowth and cessation of division when treated with dexamethasone (Guerrero et al.: Biochemical and Biophysical Research Communications 150:1185–1192, 1988). We have employed the UR61 cell line as a model for ras oncogene‐induced neuronal differentiation. In UR61 cells, dexamethasone‐induced expression of the recombinant N‐ras gene resulted in time‐dependent expression of ornithine decarboxylase enzyme (ODC) activity. Prompted by recent reports of possible functional (Lacal et al.: Molecular and Cellular Biology 7:4146–4149, 1987; Wolfman and Macara: Nature 325: 359–361, 1987) and direct (Jeng et al.: Biochemical and Biophysical Research Communications 145:782–788, 1987) interactions between oncogene ras‐coded p21 and protein kinase C (PK‐C; Ca+ +/phospholipid‐dependent protein kinase), we employed the protein kinase inhibitor H‐8 (N‐[2‐(methylamino)ethyl]‐5‐isoquinoline sulfonamide dihydrochloride) and phorbol 12, 13‐dibutyrate (PDBu) to investigate this putative interaction in the UR61 cells, where ODC activity and neurite outgrowth were used as indicators of oncogenic N‐ras action. Treatment of UR61 cells with PDBu depleted cells of PK‐C and failed to promote neurite outgrowth but enhanced N‐ras‐induced neurite outgrowth and ODC activity. H‐8, which suppressed ODC induction by forskolin and phorbol myristate acetate, enhanced both N‐ras‐induced ODC activity and neurite outgrowth. Inhibition of ODC activity by difluoromethylornithine (DFMO) did not suppress oncogenic ras‐induced neurite outgrowth, suggesting that these two ras‐triggered events are mechanistically independent. These findings suggest that certain actions of N‐ras can occur in cells depleted of PK‐C, and thus, the role of PK‐C in ras‐induced differentiation differs from its role in ras‐induced mitogenesis and transformation.
Role of Na+/H+ exchange in the granulocyte‐macrophage colony‐stimulating factor‐dependent growth of a leukemic cell line
Authors: Caracciolo Daniele , Pannocchia Antonella , Treves Silvia , Ghigo Dario , Gallo Eugenio , Tarella Corrado , Bussolino Federico , Turrini Franco , Tamponi Giacomo , Bosia Amalia
Published: February 2005

The growth of the human leukemia cell line AML‐193 in a serum‐free medium is strictly dependent on the presence of the cytokine granulocyte‐macrophage colony‐stimulating factor (GM‐CSF), which is one of the major regulators of the myelomonocytic lineage. At present, little is known about the mechanisms by which this growth factor transduces the signal intracellularly. The results of this study demonstrate that GM‐CSF needs the operation of a Na+/H+ exchanger, which is located in the plasma membrane of almost every vertebrate cell. In fact, the GM‐CSF‐dependent proliferation of AML‐193 cells is strongly reduced in the presence of the amiloride analog EIPA, a specific inhibitor of the Na+/H+ exchanger. When acidified, AML‐193 cells are able to recover the original pH, in a Na+‐dependent and EIPA‐inhibitable way; this demonstrates for the first time the presence of the Na+/H+ exchanger in these cells. Finally, GM‐CSF, at doses superimposable to those needed for triggering proliferation, induces in AML‐193 cells a sustained alkalinization, which is dependent on a operating Na+/H+ exchange, as it is inhibited by EIPA. These results suggest that GM‐CSF, like other growth factors in other cell systems, exerts its mitogenic activity in AML‐193 cells by inducing a Na+/H+ exchanger‐mediated rise in pHi.
DNA synthesis inhibition and reduced functional differentiation of midpregnant mouse mammary epithelia on collagen gels
Authors: Trumpbour Virginia , Rocha Victor
Published: February 2005

Mammary epithelial cells were examined for a link between DNA synthesis and subsequent synthesis and secretion of casein. Cells isolated from mice midway through pregnancy and cultured on collagen gels spread to form monolayers (spreading phase). Release of monolayer/gels to float in surrounding culture medium induces synthesis and secretion of casein (secretory phase). DNA synthesis was blocked during the spreading phase with cytosine arabinofuranoside (ARAC). Culture medium was assayed for casein by direct quantification of protein from SDS‐PAGE fluorographs, and by immunoblotting. When induced to become secretory, cells exposed to ARA C during the spreading phase showed a marked reduction of secretion of casein as compared to control cultures (72% reduction). In contrast, cells exposed to ARA C during the secretory phase (after monolayer formation was complete) showed no significant reduction in secretion of casein. Measurement of intracellular casein in secretory phase cells showed that reduced secretion of casein by cultures blocked during the spreading phase occurs as a consequence of reduced levels of casein synthesized, and not because of an inability to secrete intracellular accumulations. The inhibitor effect was specific; there was no significant reduction in levels of total intracellular protein synthesis, and neither cell spreading nor monolayer formation was impaired by treatments. These data support the notion that DNA synthesis is a prerequisite to functional differentiation of midpregnant mouse mammary epithelia maintained on floating collagen gels.
Mycoplasmas produce double‐stranded ribonuclease
Authors: Marcus Philip I. , Yoshida Itsuro
Published: February 2005

Mycoplasmas (Mollicutes) constitute a constant threat as insidious contaminants of animal cell cultures. They are responsible for myriad biochemical reactions associated with the cells they infect, and undoubtedly have been the source of metabolic and physiological activities attributed to their hosts. In an attempt to demonstrate a dsRNA‐inducible double‐stranded ribonuclease (dsRNase) in mammalian cells, comparable to that reported in avian cells, we discovered high levels of dsRNase “induced” by a particular stock of vesicular stomatitis virus. We now report that the double‐stranded ribonuclease resulted from the activity of a contaminant in that stock–;a “noncultivable” Mycoplasma hyorhinis. This report demonstrates the ubiquitous distribution of dsRNase among mycoplasmas, presents some characteristics of the enzyme and its production, and implicates once again mycoplasmas as contaminants of cell culture and potential perturbers of cellular physiology.
HSP 70 gene expression in Trypanosoma cruzi is regulated at different levels
Authors: de Carvalho Elizeu F. , de Castro F. T. , Rondinelli E. , Soares Celia M. A. , Carvalho Jose Francisco
Published: February 2005

The level of HSP 70 mRNA is altered in Trypanosoma cruzi cells incubated at supra‐optimal temperatures: the total amount of this RNA per cell is increased at 37°C, and slightly decreased at 40°C relative to its level at 29°C. However, its amount is greater in the polysomes at either temperature. The relative increase of this RNA is larger in the polysomes fraction than it is in the total RNA. In addition the level of HSP 70 protein in heat‐shocked cells is greater than would be expected from the recruitment of HSP 70 mRNA in the polysomal fraction. Taken together the data are interpreted as indicating that at 37°C and 40°C the HSP 70 gene regulation in T. cruzi involves both the selective accumulation of the HSP 70 mRNA in the polysomes and its preferential translation. At 37°C, in addition, an increase in the total amount of this template is observed in the cells.
Novel ATP‐binding heat‐inducible protein of Mr = 37,000 that is sensitive to transformation in BALB/3T3 cells
Authors: Nakai Akira , Hirayama Chisato , Ohtsuka Kenzo , Hirayoshi Kazunori , Nagata Kazuhiro
Published: February 2005

Using affinity chromatography on ATP‐agarose, we have identified a major ATP‐binding protein in Nonidet P‐40 extracts of avian and mammalian cells labeled with [35S]methionine. After washing ATP‐agarose beads with high‐ionic‐strength buffer (0.4 M NaCl), the 37‐kD protein was shown to be one of the major ATP‐binding proteins while p72 and grp78, which are members of the hsp70 family, also bound to ATP‐agarose. This protein consisted of several spots on two‐dimensional gel electrophoresis. The isoelectric point of the most basic spot was approximately 9.2 in chick embryo fibroblasts, whereas it was about 8.8 in mouse 3T3 cells. The identities of these proteins in mouse and chick cells were confirmed by peptide mapping. After heat‐shock treatment of BALB/3T3 cells, the major heat‐shock protein, hsp70, was shown to be induced very rapidly after heat shock and was recovered in the ATP‐binding fraction. Besides hsp70, a 37‐kD protein was also found to be induced by heat shock. This protein was drastically induced by treating the cells with α,α′‐dipyridyl, an iron chelating reagent, but not with sodium arsenite, calcium ionophore, or tunicamycin. The synthesis and the total amount of this ATP‐binding protein increased in mouse 3T3 cells transformed by simian virus 40, methylcholanthrene, or activated c‐Ha‐ras oncogene compared to their normal counterparts. The incorporation of [32P]orthophosphate was not detected in either normal or transformed cells. These studies established that a major ATP‐binding protein of Mr = 37,000 is a heat‐inducible protein and that the synthesis of this protein is regulated by malignant transformation.
Ca‐dependent slow action potentials in neuromuscular diseases
Authors: Siri L. Nicola , Dubrovsky A. L. , Uchitel O. D.
Published: February 2005

Slow Ca‐dependent action potentials were studied in skeletal muscle fibers from different Neuromuscular Diseases (NMD). Byopsies were obtained from: 3 my‐opathies [Fascioscapulohumeral Dystrophy (FSH) and Polymyositis (PM)], 6 patients with other diseases (CD) [Amyotrophic Lateral Sclerosis (ALS), Central Core Disease, Mitochondrial Myopathy, Polyneuritis (PN), von Eulenberg's Paramyotonia], and 8 normal control muscles. Experiments were carried out in muscle fibers under current‐clamp conditions. Membrane currents other than Ca ones were abolished or greatly diminished. Muscle fibers produced any of 3 types of responses, when stimulated by depolarizing pulses: fully developed Ca‐action potentials (CaAP), abortive non‐regenerative Ca responses (NrR), or only capacitive passive responses (WR). The 3 types of responses were not dependent on the basal conditions of the fibers. The frequency of observation of CaAPs was significantly higher in myopathic disease. In myopathies, 46% of the muscle fibers had CaAPs, while only 22% of fibers from CD and 15% of the fibers from normal muscles showed CaAPs. No differences were observed in the resting constants as well as in the CaAPs parameters between normal and diseased muscle fibers.
Altered cell cycle responses to insulin‐like growth factor I, but not platelet‐derived growth factor and epidermal growth factor, in senescing human fibroblasts
Authors: Chen Yuchyau , Rabinovitch Peter S.
Published: February 2005

Human diploid fibroblasts (HDF) were used to study aging‐related changes in the proliferative response to platelet‐derived growth factor (PDGF), epidermal growth factor (EGF), and insulin‐like growth factor I (IGF‐I, somatomedin‐C) in serum‐free, chemically defined culture medium. Cell cycle kinetic parameters were determined by using 5‐bromodeoxyuridine incorporation and flow cytometric analysis with the DNA stain Hoechst 33258. This allowed analysis of the growth factor response tobe focussed exclusively upon of the cycling faction of cells within the culture, even in senescent cell cultures which contained predominantly nondividing cells. PDGF and EGF exert their primary effect upon regulation of the proportion of cycling cells in the culture. The doses of PDGF and EGF that produced a half‐maximal cycling fraction, analogous to Km, showed no large or consistent difference between young‐ and old‐passage cells. In contrast, IGF‐I primarily affects the rate of transition of cells from G1, into S phase, and the dose of IGF‐I which produced a half‐maximal rate of G1 exit increased up to 130‐fold in older‐passage cells. Unexpectedly, supraPhysiologic concentrations of IGF‐I were found to increase the G1, exit rate of the dividing subpopulation of cells in older‐passage cultures to rates higher than those seen in young cultures. In summary, among cells capable of cycling in aging cultures, there were few changes in the regulation of the growth fraction by PDGF and EGF, but there was a greatly increased dependence on IGF‐I for regulation of the rate of entry into S phase. The slower growth of the dividing population of cells in aging cultures may be related to a requirement for IGF‐I at levels which are greatly above those usually supplied.