bluuebunny/crossref_metadata_2025_split huggingface dataset:
Diacylglycerol and calcium induce rapid enhancement of A‐system amino acid transport by independent mechanisms in human T‐lymphocytes
Authors: Woodlock Timothy J. , Segel George B. , Lichtman Marshall A. 
Published: February 2005

AbstractSn‐1,2‐diacylglycerols (DAG) and ionized‐free calcium can act as intracellular second messengers for cell activation. Traditionally, T‐lymphocyte activation is assessed by measurements of DNA synthesis or lymphokine production, but these responses require several days to occur and involve multiple intermediary regulatory steps. In contrast, we have found that T‐lymphocytes demonstrate rapid enhancement of A‐(alanine‐favoring) system amino acid uptake when treated with DAG or ionomycin. A 30–40% increase in the initial velocity of uptake (vi) of the synthetic A‐system specific amino acid, methylamino‐isobutyric acid (MeAIB), was measured following 5 min of exposure to DAG or ionomycin. The vi was enhanced 60% from 12 to 19 μmol/liter cell water per min after 30 min exposure of T‐cells to optimal concentrations of dioctanoylglycerol (30 μM), oleoylacetylglycerol (30 μM), or ionomycin (5 μM) (P < .01 for each agent). A 50‐fold excess of non‐radioactive MeAIB inhibited 80% of [14C]MeAIB uptake in both unstimulated and stimulated cells, indicating that uptake remained largely carrier‐mediated on treatment with these agents. Cycloheximide, 100 μg/ml, inhibited protein synthesis but did not block the A‐system amino acid transport enhancement induced by DAG or ionomycin. The DAG‐induced increase in the vi was blocked 40% with 100 μM H‐7, an inhibitor of protein kinase C. H‐7 treatment did not inhibit the ionomycin‐induced A‐system enhancement. A marked increase in cytoplasmic free calcium was measured when T‐lymphocytes were exposed to ionomycin but not on DAG exposure, and the A‐system effect of ionomycin but not DAG was blocked by extracellular EGTA. These data are compatible with two pathways for rapid enhancement of A‐system amino acid uptake in T‐lymphocytes. DAG stimulation is mediated via protein kinase C whereas ionomycin produces an A‐system effect of similar magnitude independent of protein kinase C by an increase in cytoplasmic calcium.
Functional receptors for nerve growth factor on Ewing's sarcoma and Wilm's tumor cells
Authors: Thomson Timothy M. , Pellicer Angel , Greene Lloyd A. 
Published: February 2005

AbstractQuantification of changes in levels of c‐fos RNA was used as an indicator of the presence of functional responses to nerve growth factor in several human nonneuronal cell lines which have previously been shown to express high levels of NGF receptors. Four Ewing's sarcomas, one Wilm's tumor, and one melanoma were examined. Of these cell lines, the Ewing's sarcoma IARC‐EW1 showed greatly increased levels (10–20‐fold) of c‐fos RNA after 1 hour of exposure to NGF. Except for the melanoma line, the other tumor lines exhibited small, but reproducible, elevation of c‐fos RNA expression. In IARC‐EW1 cells, this induction was analyzed for kinetics, dose‐response, and suppression by selective inhibitors of NGF action. The results indicate that these cells bear high‐affinity receptors for NGF, which utilize signal pathways similar to NGF receptors on PC12 cells. Thus, we report new types of cells with functional responses to NGF and indicate that these may constitute a new model which will usefully complement those presently used for studying the mechanism of action of NGF.
Transforming growth factors beta slow down cell‐cycle progression in a murine interleukin‐2 dependent T‐cell line
Authors: Stoeck Michael , Miescher Sylvia , MacDonald H. Robson , Von Fliedner Vladimir 
Published: February 2005

AbstractTransforming growth factors beta (TGF‐β) inhibit the growth of a variety of cell types, including lymphocytes. The immunosuppressive effects of TGF‐β have been attributed to the interference of these molecules with the interleukin‐2 (IL2)‐driven component of lymphocyte proliferation. In order to elucidate in more detail the effects of TGF‐β on IL‐2‐induced proliferation, we investigated the effects of porcine transforming growth factor beta 1 and 2 (pTGF‐β1 and 2) on the IL‐2‐driven proliferation of a murine IL‐2‐dependent T‐lymphocyte line (CTLL). The results showed that pTGF‐β1 and 2 decreased 3H‐thymidine incorporation in CTLL cells in a dose‐dependent fashion (maximum decrease of 75–85%). Combined‐time kinetic analysis of the effects of pTGF‐β on 3H‐thymidine incorporation, cell growth, and cell‐cycle distribution (monitored as DNA content distribution) revealed that, in the first 48 h of culture, pTGF‐β1 increased the doubling time from 11.4 to 19.2 h without significantly affecting the cell‐cycle distribution of CTLL cells. After 96 h of culture in the presence of pTGF‐β1, cells started to accumulate in G0/G1, although at this time point 30% of the pTGF‐β1‐treated cells were still in S‐G2/M. Furthermore, during the first 48 h, neither the expression of the 55 kd chain of the IL‐2 receptor (IL‐2R) nor the expression of the transferrin receptor (TfR) was affected by TGF‐β. After 72 h of culture in the presence of pTGF‐β1, the expression of the IL‐2R and TfR was decreased. The data suggest that in CTLL cells TGF‐β initially slows the progression of cells in all phases of cell cycle. In addition, the initial TGF‐β‐mediated decrease of IL‐2‐induced 3H‐thymidine incorporation and cell proliferation in CTLL cells is not due primarily to downregulation of the IL‐2R and/or TfR.
Transformed cells overexpress a cytosolic nucleic acid binding protein
Authors: Stoler Daniel L. , Manly Kenneth F. 
Published: February 2005

AbstractA protein blotting technique was used to identify a 57,000 dalton cytosolic nucleic‐acid‐binding protein found in neoplastically transformed cell lines. Specifically, greater amounts of this protein were found in Kirsten Murine Sarcoma Virus‐, Simian Virus 40‐, and methylcholanthrene‐transformed Balb 3T3 cells than in comparable untransformed cells. An analogous protein was identified in other transformed mammalian cells. Increased levels of the DNA binding protein in sarcoma virus transformants were shown to be dependent on the continued maintenance of the transformed phenotype. The properties of this protein are compared to those of other previously reported nucleic acid binding proteins.
The Na+, K+, 2Cl−‐cotransport system in HeLa cells and HeLa cell mutants exhibiting an altered efflux pathway
Authors: Kort Jens J. , Koch Gebhard 
Published: February 2005

AbstractWe have investigated the characteristics of a transport system in HeLa cells, which turned out to be very similar to a previously described Na+, K+, 2Cl−‐cotransport system. For further understanding about the physiological role of the cotransporter, we have mutagenized HeLa cells and selected progeny cells for growth in low potassium (0.2 mM) medium. The selected HeLa cells (LK1) exhibited alterations in the Na+, K+, 2Cl−‐cotransport system. LK, cells showed a remarkable reduction of 86Rb+ efflux via the cotransporter when compared to the parental HeLa cells. In contrast, bumetanide‐sensitive potassium influx, measured by 86Rb+ uptake, was increased in the LK1, cells (increase in vmax). Km values of the cotransporter in HeLa cells and LK1 mutants revealed similar properties for 86Rb+ and 22Na+ uptake. In addition, (3H)‐bumetanide binding studies were carried out on intact HeLa cells; 1.7 pmol/mg protein (3H)‐bumetanide was specifically bound to HeLa parental cells, which could be calculated to a number of 103,000 binding sites/cell. LK1cells present, 1.44 pmol/mg protein, specifically bound (3H)‐bumetanide and, respectively, 137,000 binding sites/cell. The LK1 cells also exhibited an increase in the number of (3H)‐ouabain binding sites as well as an increase in the activity of the Na+, K+‐ATPase, expressed as a function of ouabain‐sensitive86 Rb+ uptake. Furthermore, LK1cells were different in the concentrations of intracellular Na+ (↑)and K+ (↓) when compared to the HeLa parental cells. When grown in low K+ medium (0.2 mM K+), protein content and cell volume were increased in the LK1 cells, while the DNA content was not significantly different between both cell lines.
Expression of Na,K‐ATPase alpha subunit isoforms in the human ciliary body and cultured ciliary epithelial cells
Authors: Martin‐Vasallo Pablo , Ghosh Sikha , Coca‐Prados Miguel 
Published: February 2005

AbstractWe have analyzed the expression of Na, K‐ATPase alpha subunit isoforms in the transporting ciliary processes of the human eye and in cultured cells derived from non‐pigmented (NPE) and pigmented (PE) ciliary epithelium. Northern hybridization analysis shows that the mRNAs encoding all the three distinct forms of Na, K‐ATPase alpha subunit [alpha 1, alpha 2, and alpha 3] are expressed in the human ciliary processes in vivo. Immunohistochemical analysis using antibodies specific for each of the three alpha subunit isoforms confirms that these polypeptides are present in the microsomal fraction from the human ciliary processes. The monoclonal antibody McB2, which is specific to the Na, K‐ATPase alpha 2 subunit isoform, has been found to decorate specifically the basolateral membrane domains of NPE cells but not of the PE cells, suggesting its expression in vivo only in the ocular NPE ciliary epithelium. However, cultured cells derived from the NPE and PE layers exhibit a different pattern of expression of mRNA and protein for the Na, K‐ATPase alpha subunit isoforms when compared to the tissue. Both the NPE and PE cells express alpha 1 and alpha 3 mRNA and polypeptide, whereas alpha 2 mRNA and polypeptide are undetectable in these cells. The established cell lines derived from the NPE layer express comparable levels of the alpha 1 and alpha 3 isoforms of Na, K‐ATPase as detected in the primary culture. However, the established NPE cell lines are also distinguishable from the normal PE cells when analyzed by Western blot analysis with A × 2 antibodies. The results presented here clearly show that the NPE and PE cells in the ciliary body have a distinct expression of Na, K‐ATPase alpha subunit isoforms as compared to cultured cells.
Effects of oxygen‐free radicals on proliferation kinetics of cultured rabbit articular chondrocytes
Authors: Vincent FrançOise , Brun Hervé , Clain Elisabeth , Ronot Xavier , Adolphe Monique 
Published: February 2005

AbstractIt can be postulated that among the factors implicated in cartilaginous lesions, oxygen‐derived free radicals seem to have a prominent part. To investigate this hypothesis, rabbit articular chondrocyte cultures have been exposed to oxygenderived reactive species generated by the hypoxanthine–xanthine oxydase system. We observed a dose‐dependent decrease of cellular growth. In order to explain this result, cell cycle progression and binucleate cell fractions have been studied. A greater number of binucleate cells and an increase in cell volume were observed. Flow cytometry analysis revealed a perturbation in cell cycle progression leading to a significant increase in the proportion of cells in G2 phase and an important augmentation in cell protein content confirmed by biochemical assays. This model shows which type of alteration can be induced by oxygen‐derived free radicals in vitro. In addition, we deem this model to be useful for studying degenerative processes and for screening drugs that can scavenge oxygen‐free radicals.
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

AbstractMammary 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

AbstractSynthesis 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

AbstractThe 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

AbstractGrowth 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

AbstractMicroscopic 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

AbstractIt 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

AbstractCultures 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.