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  • 1
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of intracellular pH (pHin) in the regulation of cell growth in both normal and transformed cells is a topic of considerable controversy. In an effort to study this relationship NIH 3T3 cells were stably transfected with the gene for the yeast H+-ATPase, constitutively elevating their pHin. The resulting cell line, RN1a, has a transformed phenotype: The cells are serum independent for growth, clone in soft agar, and form tumors in nude mice. In the present study, we further characterize this system in order to understand how transfection with this proton pump leads to serum-independent growth, using defined media to investigate the effects of specific growth factors on the transfected and parental NIH 3T3 cells. While both cell lines show similar growth increases in response to platelet-derived growth factor (PDGF)-BB and epidermal growth factor (EGF), they respond differently to insulin, insulin-like growth factor-I (IGF-I) and PDGF-AA. RN1a cells exhibit increased growth at nanomolar concentrations of insulin but the parental cells had only a relatively minor response to insulin at 10 μM. Both cell lines showed some response to IGF-I in the nanomolar range but the response of RN1a cells was much larger. Differences in insulin and IGF-I receptor number alone could not explain these results. The two cell lines also respond differently to PDGF-AA. RN1a cells are relatively insensitive to stimulation by PDGF-AA and express fewer PDGF α receptors as shown by Northern blots and receptor-binding studies. We propose a unifying hypothesis in which the H+-ATPase activates a downstream element in the PDGF-AA signal transduction pathway that complements insulin and IGF-I signals, while leading to downregulation of the PDGF α receptor. © 1994 wiley-Liss, Inc.
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  • 2
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: NIH-3T3 cells transfected with yeast H+-ATPases (RN1A cells) are tumorigenic (Perona and Serrano, 1988, Nature, 334: 438). We have previously shown that RN1a cells maintain a chronically high intracellular pH (pHin) under physiological conditions. We have alsoshown that RN1a cells are serum-independent for growth, maintain a higher intracellular Ca2+(in), and glycolyze more rapidly than their non-transformed counterparts (Gillies et al., Proc. Natl. Acad. Sci., 1990, 87: 7414; Gillies et al., Cell. Physiol. Biochem., 1992, 2: 159). The present study was aimed to understand the interrelationships between glycolysis, pHin, and [Ca2+]in in RN1a cells and their non-transformed counterparts, NIH-3T3 cells. Our data show that the higher rate of glycolysis observed in RN1a cell is due to the presence of low affinity glucose transporters. Consequently, the higher rate of glycolysis is exacerbated at high glucose concentration in RN1a cells. Moreover, the maximal velocity (Vmax) for glucose utilization is up to sixfold higher in RN1a cells than in the NIH-3T3 cells, suggesting that the number of glucose transporters is higher in RN1a than NIH-3T3 cells. Glucose addition to NIH-3T3 cells results in modest decreases in both pHin and [Ca2+]in. In contrast, RN1a cells respond to glucose with a large decrease in pHin, followed by a large decrease in [Ca2+]in. The decrease in [Ca2+]in observed upon glucose addition is likely due to activation of Ca2+-ATPase by glycolysis, since the Ca2+ decrease is abolished by the Ca2+ ATPase inhibitors thapsigargin and cyclopiazonic acid. Glucose addition to ATP-depleted cells results in a decrease in [Ca2+]in, suggesting that ATP furnished by glycolysis is utilized by this pump. © 1994 Wiley-Liss, Inc.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 11 (1995), S. 137-144 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; HXK1 ; HXK2 ; GLK1 ; mRNA ; transcriptional control ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In Saccharomyces cerevisiae, the transcriptional regulation of most glycolytic genes has been extensively studied. By contrast, little is known about the transcriptional control of the three glucose-phosphorylating enzymes, although this catalytic reaction has an important role in the regulation of cell metabolism. In this paper, we describe the transcriptional regulation of the HXK1, HXK2 and GLK1 genes in the hope of revealing differences in the steady-state levels of mRNA associated with a particular carbon source used in the culture medium. Our results provide evidence supporting a differential expression of the three genes depending on the carbon source used for growth. We have also studied the induction and repression kinetics of mRNA expression for the HXK1, HXK2 and GLK1 genes.
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  • 4
    ISSN: 0741-0581
    Keywords: BrdU incorporation ; Cultured cells DNA replication ; Electron microscopy ; EM immunocytochemistry ; Immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minues. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.
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  • 5
    ISSN: 0741-0581
    Keywords: Acetone ; Methanol ; Diethyl ether ; Freezing artifact ; Chemical fixation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Pollen grains of Lolium perenne (rye grass) were prepared for transmission electron microscopy by rapid freezing in liquid propane, substitution in acetone, methanol or diethyl ether, and embedment in the acrylic resin London Resin gold. These were compared to pollen chemically fixed (CF) in aldehyde/osmium tetroxide and embedded in the epoxy resin Quetol 651. Ultrastructural preservation was superior in freeze-substituted (FS) pollen, particularly with the use of acetone or methanol. Optimally preserved FS pollen displayed a homogeneous aspect of the cytoplasm and nucleoplasm, and smooth, uninterrupted contour or organelles. A striking difference was also seen in the preservation of inclusions in the intine. Varied forms and sizes of intine inclusions were evident in FS pollen but these were not discernible in the CF image. The FS scheme studied here presents enormous potential for both ultrastructural and immunolabelling studies in rye grass pollen. Problems discussed include artifacts associated with each of the substitution solvents used, and a gradient of freezing damage observed within the pollen grain.
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  • 6
    ISSN: 0749-503X
    Keywords: Candida albicans ; heat-shock (stress) proteins ; nucleotide sequence ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: We have cloned and sequenced a Candida albicans gene (SSB1) encoding a potential member of the heat-shock protein seventy (hsp70) family. The protein encoded by this gene contains 613 amino acids and shows a high degree (85%) of sequence identity to the ssb subfamily (ssb1 and ssb2) of the Saccharomyces cerevisiae hsp70 family. The transcribed mRNA (2·1 kb) is present in similar amounts both in yeast and germ tube cells of C. albicans. The sequence data has been deposited in the GenBank data library under the Accession Number X97723. © John Wiley & Sons, Ltd.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 14 (1998), S. 501-515 
    ISSN: 0749-503X
    Keywords: Saccharomyces cerevisiae ; bakers' yeast ; pH homeostasis ; cytoplasmic pH ; vacuolar pH ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: The effects of monovalent cations on the internal pH of yeast were studied. Our former procedure was modified, inducing maximal alkalinization of the cells with 100 mM-NH4OH instead of Tris base. The pH values were lower than reported before (Peña et al., J. Bacteriol. 1995 177, 1017-1022). With glucose as substrate, the internal cytoplasmic pH reached higher values when incubating at an external pH of 6·0, as compared to pH 4·0. Monovalent cations added approximately 5 min after glucose produced a further increase in the internal pH, which was higher at a previous incubation pH of 4·0 than that observed at pH 6·0. The selectivity of the changes followed a similar order to that of the transport system for monovalent cations.When incubating cells with glucose for more than 30 min, the initial changes of the internal pH appeared to be regulated by the cell. However, under the fluorescence microscope, it was observed that pyranine, which was confined to the cytoplasm during the first 15 min, was progressively concentrated in the vacuole. By studying the fluorescence changes of cells electroporated and then incubated with glucose or glucose plus potassium, we could follow the internal pH of this organelle, obtaining values within the range reported by other authors. Also, in cells preincubated with glucose for 60 min, and electroporated afterwards, the fluorescence of pyranine, which only entered the cytoplasm, allowed us to measure the pH of this compartment, showing that it was more alkaline than the vacuole. Moreover, the cytoplasmic pH increased upon addition of glucose or potassium. The vacuolar pH, on the other hand, increased upon addition of potassium after glucose, but decreased upon addition of glucose. In addition, incubation of the cells with glucose with or without pyranine produced vesiculation of the vacuole. © 1998 John Wiley & Sons, Ltd.
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  • 8
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The morphology of cells and the organization of axons were studied in Golgi-Colonnier and toluidine blue stained preparations from the medial cerebral cortex of the lizard Lacerta pityusensis. In the medial cortex, six strata were distinguished between the superficial glial membrane and the ependyma. Strata I and II formed the outer plexiform layer, stratum III formed the cellular layer, and strata IV go VI the inner plexiform layer. The outer plexiform layer contained smooth bipolar neurons; their dendrites were oriented anteroposteriorly and their axons were directed towards the posterior zone of the brain. Five neuronal types were observed in the cellular layer. The spinous pyramidal neurons had well-developed apical dendrites and poorly developed basal ones. Their axons entered the inner plexiform layer and gave off collaterals oriented anteroposteriorly. The small, sparsely spinous pyramidal neurons had poorly developed dendrites and their axons entered the inner plexiform layer. The spinous bitufted neurons had well-developed apical and basal dendritic tufts. Their axons gave off collaterals that reached the outer and inner plexiform layers of both the dorsomedial and dorsal cortices. The sparsely spinous horizontal neurons had dendrites restricted to the outer plexiform layer. Their axons entered the inner plexiform layer. The sparsely spinous, multipolar neurons had their soma close to stratum IV and their axons entered the outer plexiform layer. In stratum V of the inner plexiform layer were large, spiny polymorphic neurons; they had dendrites with long spines, and their axons reached the cellular layer. On the basis of these results, we have subdivided the medial cortex into two subregions: the superficial region, which contains the neurons of the cellular layer and their dendritic domains, and the deep region, strata V and VI, which contains the large, spiny polymorphic neurons. The neurons in the medial cortex of these lizards resembles those in the area dentata of mammals. On this basis, the superficial region may be compared to the dentate gyrus and the deep region to the hilar region of the hippocampus of mammals.
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  • 9
    ISSN: 0730-2312
    Keywords: osteoblasts ; insulin-like growth factor-I ; calcium signaling ; fura 2 ; digital imaging ; receptor crosslinking ; Northern analysis ; Scatchard analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The role of the IGF-II/cation independent mannose-6-phosphate (IGF-II/M6P) receptor in the transduction of cellular effects evoked by IGF-II has been extensively debated in the literature. Many reports suggest that IGF-II transduces its effects through the IGF-I receptor, while others show that IGF-II utilizes the type II receptor to affect cellular activity. This study (1) verifies the presence of the IGF-II/M6P receptor in rat calvarial osteoblasts, and (2) evaluates the ability of the receptor to initiate intracellular single. Using conventional receptor binding assays, it was found that osteoblasts bind IGF-II with high affinity. Scatchard analyses indicated that there are 5.08 × 104 IGF-II/M6P receptor per osteoblast with a Kd near (2.0 nM). The receptor protein was further identified by cross-linking with 125I-IGF-II. Northern analysis was used to identify an mRNA transcript for the IGF-II/M6P receptor protein. To examine if the IGF-II/M6P receptor can initiate second messenger signals, the ability of IGF-II to evoke Ca2+ transients was evaluated. Osteoblasts pretreated with IGF-I did not lose their ability to respond to IGF-II. Further, a polyclonal antibody against the rat IGF-II/M6P receptor (R-II-PAB1) (1) was able to evoke its own Ca2+ response, and (2) was able to block the generation of Ca2+ transients caused by IGF-II. The data in this report show that the osteoblastic Ca2+ response to IGF-II appears to be caused by an intracellular release of Ca2+ which is mediated by the IGF-II/M6P receptor making it possible to envision how the receptor may be an important modulator of osteoblast mediated osteogenesis. © 1995 Wiley-Liss, Inc.
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  • 10
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two types of choanocyte-like cells have been found in the digestive tract of the starfish. Type I choanocytes are in the lining epithelium of all organs of the digestive system. These are narrow, columnar cells strongly anchored basally and expanded apically into a protuberance projecting into the lumen. A prominent flagellum surrounded by microvilli projects from the center of this protuberance. Apical cytoplasm contains numerous mitochondria, secondary lysosomes, and multivesicular bodies. A distinctive characteristic of these cells is a filament bundle that traverses the length of the cell from its region of attachment on the rootlet of the flagellar basal body to its terminus on the basal plasma membrane. Between the attenuated basal ends of type I cells are the nerve fibers of an intraepithelial nerve plexus. Thickness of the plexus is correlated with the quantity of type I cells in the epithelium.Type II choanocytes are in the cuboidal coelomic epithelium that forms the outer layer of digestive tract organs. These cells are smaller than those of type I, and they have an apical collar surmounted by a ring of 13 microvilli. Within the collar is a cup-shaped depression with a central flagellum. Coated vesicles, secondary lysosomes, and phagocytic infoldings are observed in and near the collar cytoplasm. Filament bundles similar to those in type I choanocytes are also observed in coelomic epithelial cells that are sufficiently tall. Injection of peroxidase into the stomach and ferritin into the coelom results in phagocytic uptake of these macromolecules by type I and type II choanocytes, respectively.
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