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  • 1
    Electronic Resource
    Electronic Resource
    Assembly and bundling of marginal band microtubule protein: Role of tau (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 29 (1994), S. 57-71 
    Details
    ISSN: 0886-1544
    Keywords: microtubule bundling ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule protein extracted from dogfish erythrocyte cytoskeletons by disassembly of marginal bands at low temperature formed linear microtubule (MT) bundles upon reassembly at 22°C. The bundles, which were readily visible by video-enhanced phase contrast or DIC microscopy, increased in length and thickness with time. At steady state after 1 hour, most bundles were 6-11 μm in length and 2-5 MTs in thickness. No inter-MT cross-bridges were visible by negative staining. The bundles exhibited mechanical stability in flow as well as flexibility, in this respect resembling native marginal bands. As analyzed by SDS-PAGE and immunoblotting, our standard extraction conditions yielded MT protein preparations and bundles containing tau protein but not high molecular weight MAPs such as MAP-2 or syncolin. In addition, late fractions of MT protein obtained by gel filtration were devoid of high molecular weight proteins but still produced MT bundles. The marginal band tau was salt-extractable and heat-stable, bound antibodies to mammalian brain tau, and formed aggregates upon desalting. Antibodies to tau blocked MT assembly, but both assembly and bundling occurred in the presence of antibodies to actin or syncolin. The MTs were “unbundled” by subtilisin or by high salt (0.5-1 M KCl or NaCl), consistent with tau involvement in bundling. High salt extracts retained bundling activity, and salt-induced unbundling was reversible with desalting. However, reversibility was observed only after salt-induced MT disassembly had occurred. Reconstitution experiments showed that addition of marginal band tau to preassembled MTs did not produce bundles, whereas tau presence during MT reassembly did yield bundles. Thus, in this system, tau appears to play a role in both MT assembly and bundling, serving in the latter function as a coassembly factor. © 1994 Wiley-Liss, Inc.
    Additional Material: 15 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970290106
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  • 2
    Electronic Resource
    Electronic Resource
    Localization of tau and other proteins of isolated marginal bands (1994)
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 27 (1994), S. 350-360 
    Details
    ISSN: 0886-1544
    Keywords: microtubules ; MAPs ; cytoskeleton ; tau ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: To determine which proteins were associated with and intrinsic to the marginal band (MB) of microtubules (MTs), we studied protein components of MBs isolated from nucleated erythrocytes by differential detergent solubilization of the membrane skeleton (MS). MBs isolated from dogfish erythrocytes contained major proteins in the tubulin Mr range. A high molecular weight protein of ∼290 kD that bound antibody to syncolin and to heat-stable brain MAPs was present in the whole cytoskeleton. However, most of it was solubilized by the MB isolation medium, together with the MS. Dogfish erythrocyte cytoskeletons and isolated MBs were examined with polyclonal and monoclonal antibodies against mammalian brain tau and chicken erythrocyte tau. As shown by immunofluorescence and immunoblotting, these antibodies bound to proteins in the 50 to 67 kD range, located along the length of isolated MBs. Two-dimensional SDS-PAGE revealed isolated MB proteins of pI ∼6.8 in the same molecular weight range, as well as α- and β-tubulin with pI ∼5.4. Subtilisin or high-salt treatment of isolated MBs resulted in unbundling of MTs, indicating involvement of MAPs. MBs isolated from chicken erythrocyte cytoskeletons also contained tau as shown by antimammalian brain tau immunofluorescence. Both chicken and dogfish isolated MBs also bound phalloidin, but the binding was usually discontinuous and, for any given MB, matched the pattern of anti-syncolin binding. Both syncolin and F-actin were part of the MS remnant remaining after MT disassembly, supporting their assignment to a specialized MS region at the MB/MS interface. In contrast, tau protein appears to be intrinsic to the MB, where it may have an MT stabilizing and bundling function. © 1994 Wiley-Liss, Inc.
    Additional Material: 13 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/cm.970270407
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  • 3
    Electronic Resource
    Electronic Resource
    Effects of extremely-law-frequency electromagnetic fields on ion transport in several mammalian cells (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Bioelectromagnetics 15 (1994), S. 579-588 
    Details
    ISSN: 0197-8462
    Keywords: ELF ; magnetic fields ; cyclotron resonance ; calcium transport ; potassium transport ; Life and Medical Sciences ; Occupational Health and Environmental Toxicology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Physics
    Notes: We have investigated the effects of sinusoidal electromagnetic fields (EMF) on ion transport (Ca2+, Na+, K+, and H+) in several cell types (red blood cells, thymocytes, Ehrlich ascites tumor cells, and HL60 and U937 human leukemia cells). The effects on the uptake of radioactive tracers as well as on the cytosolic Ca2+ concentration ([Ca2+]i), the intracellular pH (pHi), and the transmembrane potentsial (TMP) were studied. Exposure to EMF at 50 Hz and 100-2000 μT (rms) had no significant effects on any of these parameters. Exposure to EMF of 20-1200 μT (rms) at the estimated cyclotron magnetic resonance frequencies for the respective ions had no significant effects except for a 12-32% increase of the uptake of 42K within a window at 14.5-15.5 Hz and 100-200 μT (rms), which was found in U937 and Ehrlich cells but not in the other cell types. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/bem.2250150611
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  • 4
    Electronic Resource
    Electronic Resource
    Taurine release associated to volume regulation in rabbit lymphocytes (1991)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 45 (1991), S. 207-212 
    Details
    ISSN: 0730-2312
    Keywords: hyposmolarity ; swelling ; free amino acids ; DIDS ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Rabbit lymphocytes exposed to hyposmotic media first swell and then recover their initial volume within 6 min. During volume recovery, free amino acids (FAA) decrease from 451.1 to 208 nmoles/mg protein. Taurine was the dominating FAA, accounting for 70% of the FAA pool. The time course of 3H-taurine release induced by hyposmolarity followed that of volume recovery. Efflux of 3H-taurine in an 8 min period was 17.8% (of total labeled taurine accumulated during loading) in an isosmotic medium. Reducing osmolarity to 0.87, 0.75, 0.62, and 0.5 increased this release to 24.8%, 38.1%, 56.4% and 70.9%, respectively. The volume-sensitive release of 3H-taurine was unaffected by omission of external Na+ or Ca++ and was reduced by 23% in the absence of Cl-. It was unaffected by agents disrupting the cytoskeleton or by tetraethylammonium, barium, quinidine, and gadolinium, but was 26% reduced by DIDS. Taurine release was inhibited at 4°C, but was unchanged at 15°C or 25°C. An involvement of FAA, particularly taurine, in lymphocyte volume regulation is suggested.
    Additional Material: 3 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240450212
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  • 5
    Electronic Resource
    Electronic Resource
    Pancreastatin activates pertussis toxin-sensitive guanylate cyclase and pertussis toxin-insensitive phospholipase C in rat liver membranes (1994)
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 55 (1994), S. 173-181 
    Details
    ISSN: 0730-2312
    Keywords: phosphatydilinositol 1,4,5-triphosphate ; cGMP ; peptide ; hepatocyte ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have recently found the calcium dependent glycogenolytic effect of pancreastatin on rat hepatocytes and the mobilization of intracellular calcium. To further investigate the mechanism of action of pancreastatin on liver we have studied its effect on guanylate cyclase, adenylate cyclase, and phospholipase C, and we have explored the possible involvement of GTP binding proteins by measuring GTPase activity as well as the effect of pertussis toxin treatment of plasma liver membranes on the pancreastatin stimulated GTPase activity and the production of cyclic GMP and myo-inositol 1,4,5-triphosphate.Pancreastatin stimulated GTPase activity of rat liver membranes about 25% over basal. The concentration dependency curve showed that maximal stimulation was achieved at 10-7 M pancreastatin (EC50 = 3 nM). This stimulation was partially inhibited by treatment of the membranes with pertussis toxin. The effect of pancreastatin on guanylate cyclase and phospholipase C were examined by measuring the production of cyclic GMP and myo-inositol 1,4,5-triphosphate respectively. Pancreastatin increased the basal activity of guanylate cyclase to a maximum of 2.5-fold the unstimulated activity at 30°C, in a time- and dose-dependent manner, reaching the maximal stimulation above control with 10-7 M pancreastatin at 10 min (EC50 = 0.6 nM). This effect was completely abolished when rat liver membranes had been ADP-ribosylated with pertussis toxin. On the other hand, adenylate cyclase activity was not affected by pancreastatin. Phospholipase C activity of rat liver membranes was rapidly stimulated (within 2-5 min) at 30°C by 10-7 M pancreastatin, reaching a maximum at 15 min. The dose response curve showed that with 10-7 M pancreastatin, maximal stimulation was obtained (EC50 = 3 nM). GTP (10-5 M) stimulated the membrane-bound phospholipase C as expected. However, the incubation of rat liver membranes with GTP partially inhibited the stimulation of phospholipase C activity produced by pancreastatin, whereas GTP enhanced the activation of phospholipase C by vasopressin. This inhibition by GTP was dose dependent and 10-5 M GTP obtained the maximal inhibition (about 40%). the inhibitory effect of GTP on the stimulatory effect of pancreastatin on phospholipase C activity was completely abolished when rat liver membranes had previously been ADP-ribosylated with pertussis toxin. The presence of 8-Br-cGMP mimics the effect of GTP, whereas GMP-PNP increased both basal and pancreastatin-stimulated phospholipase C, suggesting a role of the cyclic GMP as a feed-back regulator of the synthesis of myo-inositol 1,4,5-triphosphate. However, the pretreatment of membranes with pertussis toxin did not modify the production of myo-Inositol 1,4,5-triphosphate stimulated by pancreastatin.In conclusion, pancreastatin activates guanylate cyclase activity and phospholipase C involving different pathways, pertussis toxin-sensitive, and -insensitive, respectively. © 1994 Wiley-Liss, Inc.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/jcb.240550204
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  • 6
    Electronic Resource
    Electronic Resource
    Yeast exo-β-glucanases can be used as efficient and readily detectable reporter genes in Saccharomyces cerevisiae (1994)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 10 (1994), S. 747-756 
    Details
    ISSN: 0749-503X
    Keywords: β-glucanases ; Saccharomyces cerevisiae ; flow cytometry ; reporter genes ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Yeast exo-1,3-β-glucanases are secretable proteins whose function is basically trophic and may also be involved in cell wall glucan hydrolytic processes. Since fluorescein di(β-D-glucopyranoside) is a fluorogenic substrate detectable and quantifiable by flow cytometry, it was used for testing the ability of the EXG1 gene product of Saccharomyces cerevisiae and its homologous gene in Candida albicans to function as reporter genes. These open reading frames were coupled to different promoters in multicopy plasmids, and exoglucanase activity quantified at flow cytometry. Exoglucanases were found to be useful tools for the study of promoter regions in S. cerevisiae. This technique has the advantage over other reporter gene systems - such as β-galactosidase fusions - that it does not require permeabilization of yeast cells and therefore it allows the recovery of viable cells - by sorting - after flow cytometry analysis.
    Additional Material: 5 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320100606
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  • 7
    Electronic Resource
    Electronic Resource
    Flow cytometric analysis of Saccharomyces cerevisiae autolytic mutants and protoplasts (1992)
    New York, NY [u.a.] : Wiley-Blackwell
    Yeast 8 (1992), S. 39-45 
    Details
    ISSN: 0749-503X
    Keywords: Flow cytometry ; autolytic mutants ; protoplasts ; yeast ; viability assay ; Life and Medical Sciences ; Genetics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Simple methods, based on the technique of flow cytometry, have been developed for the phenotypic characterization of yeast autolytic mutants and for the analysis of the formation and regeneration of the yeast protoplasts. The expression of lytic mutations determined uptake of the fluorescent dye propidium iodide, which could be carefully monitored by flow cytometry. Mixed populations of lysed and viable cells were precisely quantified and sorted, and the technique was also applied to demonstrate protection from lysis of mutant cells with cell wall defects, in the presence of osmotic stabilizers. Protoplast formation and regeneration was monitored by analysing relative cell size; this was facilitated by the preparation of homogeneous protoplast preparations. The technique of flow cytometry proved superior to other conventional methods for these types of study.
    Additional Material: 6 Ill.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1002/yea.320080104
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