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1.
Treatment of PtK1 cells with 5 mM acrylamide for 4 hr induces reversible dephosphorylation of keratin in concert with reversible aggregation of intermediate filaments (Eckert and Yeagle, Cell Motil. Cytoskeleton 11:24-30, 1988). We have examined this phenomenon by 1) in vitro phosphorylation of isolated PtK1 keratin filaments and 2) combined treatments of PtK1 cells with both acrylamide and agents which elevate intracellular cAMP levels. PtK1 keratins were incubated in gamma-32P-ATP in the presence or absence of cAMP-dependent kinase (A-kinase) and cAMP. Levels of phosphorylation were analyzed by electrophoresis and autoradiography. Phosphorylation of keratin polypeptides (56 kD, 53 kD, 45 kD, 40 kD) occurred without added kinase, suggesting the presence of an endogenous kinase which remains with intermediate filaments in residues of Triton X-100 extracted cells. Phosphorylation levels were increased by A-kinase but not by cAMP alone, indicating the presence of cAMP-dependent phosphorylation sites in addition to sites phosphorylated by the endogenous kinase. To study the possible role of cAMP-dependent phosphorylation in acrylamide-induced aggregation of keratin filaments, we treated cells with acrylamide in the presence of 8-bromo-cAMP (brcAMP), pertussis toxin (PT), isobutylmethylxanthine (IBMX), or forskolin, which increase intracellular cAMP levels. The distribution and phosphorylation levels of keratin filaments, as well as intracellular cAMP levels, were determined for each of these treatments. In addition to aggregation and dephosphorylation of keratin filaments reported previously, treatment of cells with acrylamide alone also results in reduced levels of intracellular cAMP. 8-bromo-cAMP, IBMX, and forskolin prevent acrylamide-induced aggregation of keratin filaments and result in both normal levels of keratin phosphorylation and normal intracellular cAMP levels. PT was apparently ineffective. These observations suggest that 1) PtK1 keratins are phosphorylated by cAMP-dependent kinase and an endogenous, cAMP-independent kinase and 2) alteration of levels of cAMP-dependent phosphorylation may be involved in aggregation of keratin filaments in response to acrylamide. 相似文献
2.
A confluent PtK2 cell sheet was incised in a serum-free culture medium, at 15 min, 2 hr and 24 hr after wounding. The culture media were collected in the same way and used as conditioned media. Unwounded confluent cells were cultured in the conditioned medium for 24 hr. They showed a modification of fibronectin localization similar to that which we had previously observed in wounded confluent PtK2 cells: cells lost their normal fibronectin fibrils and were surrounded by fibronectin lace. This finding suggested that during wound healing, the cells released soluble chemical factors which could modify the fibronectin localization pattern of unwounded confluent cells. Subconfluent cells did not respond to conditioned media, showing that confluent cells and subconfluent cells had different susceptibilities. 相似文献
3.
Fluorescence staining with rhodamine phalloidin specific for F-actin was employed to examine the effects of delta-9-tetrahydrocannabinol (THC) on the distribution of microfilaments in kangaroo rat epithelial cells (PtK2) and rabbit aortic endothelial cells (RAE). PtK2 cells were more sensitive to THC treatment than RAE cells. Exposure of PtK2 cells to 10 microM THC for 2 h disrupted the microfilament network. After treatment with 20 microM THC for 2 h there was a loss of cell-to-cell contact between PtK2 cells, and at 30 microM THC, the cells started to detach from the substratum. In contrast, microfilament disorganization but not cell detachment was observed in RAE cells at THC concentrations of 80 and 100 microM. The possible mechanisms which may account for the changes in the microfilament system are discussed. 相似文献
4.
Cytoskeletal dynamics in rabbit synovial fibroblasts: I. Effects of acrylamide on intermediate filaments and microfilaments 总被引:2,自引:0,他引:2
Rabbit synovial fibroblasts respond to changes in cell shape and cytoskeletal architecture by altering specific gene expression. We have tested the ability of acrylamide, a neurotoxin that alters the distribution of intermediate filaments in cultured PtK1 cells, to induce metalloprotease expression in synovial fibroblasts. Cells treated with 2-20 mM acrylamide for 5 to 24 h underwent shape changes similar to cells treated with the tumor promoter phorbol myristate acetate. Intermediate filaments visualized with anti-vimentin antibodies did not collapse into a perinuclear cap in these rounded cells, but were still present in the extended cell processes. Unexpectedly, when actin was visualized in acrylamide-treated cells, extensive dissociation and clumping of microfilaments was observed. Concentrations of acrylamide greater than 10 mM were cytotoxic, but cells recovered completely after 24 h incubation with 5 mM acrylamide. Like other agents that alter cell shape and actin distribution in synovial fibroblasts, acrylamide also induced expression of the secreted metalloprotease collagenase. Although some recent evidence suggests that acrylamide may be able to exert its collagenase-inducing effects extracellularly, perhaps through transmembrane matrix receptors, our observation that this neurotoxin dramatically alters protein synthesis in synovial fibroblasts suggests that direct effects on cell metabolism may also play a role in acute acrylamide intoxication. 相似文献
5.
Single and double-label immunofluorescence were used to study the fibronectin (FN) and keratins (Ks) localization patterns in early wounded confluent PtK2 cells. A time-course study (0 hr, 2 hr, 6 hr and 24 hr) gives the following results: before wounding, the FN localizations of confluent cells are composed of curved and sometimes branched strands or fibrils. The Ks network is formed by radial fluorescent filaments connecting the Ks centers near the nuclei with a linear fluorescence underlying the cell membrane. Two hr after, the FN localizations are redistributed at the cell-cell contact areas. The radial Ks filaments are compacted around the nuclei, some of them delineate the cytoplasmic periphery of the wounded cells. Six hr later, the method shows redistributed FN localizations at the cell-cell contact areas. An alveolar pattern is formed enclosing each of the adjacent cells. The codetected Ks filaments are retracted around the nuclei. The underlying cell-cell contact areas are also well demonstrated. It may be noted that these areas are FN-labelled. Twenty-four hr after wounding, the FN alveolar pattern persists. The redistributed Ks filaments have some similarity to those seen before wounding. 相似文献
6.
The vimentin intermediate filament (VIMF) network is more sensitive to heat-induced disruption than either the microtubule (MT) or microfilament (MF) cytoskeletal (CSK) arrays in G1 Chinese hamster ovary (CHO) cells (Coss and Wachsberger: Radiation Research, 1987). We therefore investigated the effect of the VIMF disruptive agent, acrylamide (Eckert: European Journal of Cell Biology 37:169-174, 1985), on the heat response of synchronous CHO cells. Cells, either in the process of spreading (G1 or S phase) or in the well-spread state (S phase), were exposed to a nontoxic concentration of 5 mM acrylamide, heated, and processed for immunofluorescence microscopy 30 min or 20 hr following the heat shock. Recovery from CSK disruption was related to cell survival. CHO cells, either in the process of spreading or in the well-spread state, were sensitized to heat-induced CSK disruption and cytotoxicity by acrylamide. Recovery from CSK disruption correlated with surviving fractions of cells treated in the G1 phase but not with surviving fractions of cells treated in the S phase and was independent of the degree of cell spreading. This correlation suggests that damage to CSK structures may contribute to the death of cells treated in G1 but not necessarily to the death of cells treated in S phase. The degree of acrylamide sensitization of heat-induced CSK disruption was greater for cells exposed to acrylamide prior to spreading than for well-spread cells. Furthermore, normal spreading of cells was prevented when they were plated into medium containing acrylamide, suggesting that acrylamide interferes with the initial stages of attachment and spreading of these cells. These observations are interpreted in relation to the possible role that VIMFs, together with cortical MFs, may play in mediating cell surface focal contacts in the initial stages of cell attachment and spreading. 相似文献
7.
The assembly of intermediate filaments into a cytoplasmic network was studied by microinjecting into the nuclei and cytoplasms of PtK2 cells, plasmids that contained a full length desmin cDNA and an RSV promoter. Immunofluorescence was used to monitor the expression of desmin and its integration into the cells' vimentin intermediate filament network. We found that the expressed desmin co-localized with filaments of vimentin just as it does with fluorescently labelled desmin is microinjected into the cytoplasm of PtK2 cells. As early as two hours after microinjection of the plasmids, small discrete dots and short fragments of desmin could be detected throughout the cytoplasm of the cells. This initial distribution of desmin was superimposed on the filamentous pattern of vimentin in the cells. At 8 hours after microinjection of the plasmids, some of the desmin was present in long filaments that were coincident with vimentin filaments. By 18 hours, most of the desmin was in a filamentous network co-localizing with vimentin. There was no indication that desmin assembly began in the perinuclear region and proceeded toward the cell periphery. In some cells, excessively high levels of desmin were expressed. In these cases, overexpression led to clumping of desmin filaments as well as to an accumulation of diffusely distributed desmin protein in the center of the cells. This effect was apparent at approximately 18 hours after introduction of the plasmid. The native vimentin filaments in such cells were also aggregated around the nucleus, co-localizing with desmin. The microtubule networks in all injected cells appeared normal; microtubules were extended in typical arrays out to the periphery of the cells. 相似文献
8.
Control of microtubule nucleation and stability in Madin-Darby canine kidney cells: the occurrence of noncentrosomal, stable detyrosinated microtubules 总被引:33,自引:23,他引:10
The microtubule-nucleating activity of centrosomes was analyzed in fibroblastic (Vero) and in epithelial cells (PtK2, Madin-Darby canine kidney [MDCK]) by double-immunofluorescence labeling with anti-centrosome and antitubulin antibodies. Most of the microtubules emanated from the centrosomes in Vero cells, whereas the microtubule network of MDCK cells appeared to be noncentrosome nucleated and randomly organized. The pattern of microtubule organization in PtK2 cells was intermediate to the patterns observed in the typical fibroblastic and epithelial cells. The two centriole cylinders were tightly associated and located close to the nucleus in Vero and PtK2 cells. In MDCK cells, however, they were clearly separated and electron microscopy revealed that they nucleated only a few microtubules. The stability of centrosomal and noncentrosomal microtubules was examined by treatment of these different cell lines with various concentrations of nocodazole. 1.6 microM nocodazole induced an almost complete depolymerization of microtubules in Vero cells; some centrosome nucleated microtubules remained in PtK2 cells, while many noncentrosomal microtubules resisted that treatment in MDCK cells. Centrosomal and noncentrosomal microtubules regrew in MDCK cells with similar kinetics after release from complete disassembly by high concentrations of nocodazole (33 microM). During regrowth, centrosomal microtubules became resistant to 1.6 microM nocodazole before the noncentrosomal ones, although the latter eventually predominate. We suggest that in MDCK cells, microtubules grow and shrink as proposed by the dynamic instability model but the presence of factors prevents them from complete depolymerization. This creates seeds for reelongation that compete with nucleation off the centrosome. By using specific antibodies, we have shown that the abundant subset of nocodazole-resistant microtubules in MDCK cells contained detyrosinated alpha-tubulin (glu tubulin). On the other hand, the first microtubules to regrow after nocodazole removal contained only tyrosinated tubulin. Glu-tubulin became detectable only after 30 min of microtubule regrowth. This strongly supports the hypothesis that alpha-tubulin detyrosination occurs primarily on "long lived" microtubules and is not the cause of the stabilization process. This is also supported by the increased amount of glu-tubulin that we found in taxol-treated cells. 相似文献
9.
Reorganization of plasma membrane-associated 36 000 dalton protein upon drug-induced redistribution of cytokeratin 总被引:1,自引:0,他引:1
Epithelial PtK2 cells were used as a model to study the possible redistribution of the major tyrosine protein kinase substrate, p36, upon drug-induced reorganization of the cytokeratin network. Cells were grown on glass coverslips and exposed to cytochalasin D (CD), colcemid or a combination of the two. The cytokeratin type intermediate filaments of the cells were redistributed by treatment of the cells with colchicine and CD. Simultaneous changes in cytokeratin and in p36 antigen were observed by double-label immunofluorescence. In control PtK2 cells, p36 was distributed characteristically at the cortical cytoplasm. Neither colchicine nor CD alone was able to cause a major reorganization of cytokeratin or p36. Their combined effect resulted in formation of blebs containing abundant p36 at the cell surface and at cell-cell junctions. Actin, on the other hand, was reorganized to similar configurations by CD alone. These observations show for the first time a drug-induced redistribution of p36. The results suggest a relationship between membrane-associated p36 and the cytoskeletal fibres that terminate at the plasma membrane. 相似文献
10.
Visualization of intermediate filaments in living cells using fluorescently labeled desmin 总被引:5,自引:0,他引:5
Fluorescently labeled desmin was incorporated into intermediate filaments when microinjected into living tissue culture cells. The desmin, purified from chicken gizzard smooth muscle and labeled with the fluorescent dye iodoacetamido rhodamine, was capable of forming a network of 10-nm filaments in solution. The labeled protein associated specifically with the native vimentin filaments in permeabilized, unfixed interphase and mitotic PtK2 cells. The labeled desmin was microinjected into living, cultured embryonic skeletal myotubes, where it became incorporated in straight fibers aligned along the long axis of the myotubes. Upon exposure to nocodazole, microinjected myotubes exhibited wavy, fluorescent filament bundles around the muscle nuclei. In PtK2 cells, an epithelial cell line, injected desmin formed a filamentous network, which colocalized with the native vimentin intermediate filaments but not with the cytokeratin networks and microtubular arrays. Exposure of the injected cells to nocadazole or acrylamide caused the desmin network to collapse and form a perinuclear cap that was indistinguishable from vimentin caps in the same cells. During mitosis, labeled desmin filaments were excluded from the spindle area, forming a cage around it. The filaments were partitioned into two groups either during anaphase or at the completion of cytokinesis. In the former case, the perispindle desmin filaments appeared to be stretched into two parts by the elongating spindle. In the latter case, a continuous bundle of filaments extended along the length of the spindle and appeared to be pinched in two by the contracting cleavage furrow. In these cells, desmin filaments were present in the midbody where they gradually were removed as the desmin filament network became redistributed throughout the cytoplasm of the spreading daughter cells. 相似文献
11.
Gelatin-induced Reversion of Protoplasts of Bacillus subtilis to the Bacillary Form: Biosynthesis of Macromolecules and Wall During Successive Steps 总被引:15,自引:6,他引:9
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Protoplasts of Bacillus subtilis plated on SDG medium formed L colonies in quantative yield and propagated in the L-form indefinitely. Protoplasts or L bodies placed in 25% gelatin medium formed bacillary colonies. Details of the reversion of these naked bodies to the walled form are reported here. Protoplasts prepared in minimal medium reverted fairly synchronously 3 to 4 hr after inoculation into gelatin, but protoplasts preincubated in casein hydrolysate (CH)-enriched minimal medium were primed to revert within 1 hr in the gelatin. Preincubation for 1.5 hr in 0.44% CH was required for good priming. Cells must be subjected to this preincubation (step 1) in the naked state; it is effective for L bodies as well as protoplasts. Priming was blocked by chloramphenicol, puromycin, and actinomycin D but was not affected by penicillin, lysozyme, or inhibition of deoxyribonucleic acid (DNA) synthesis. It is concluded that protein and ribonucleic acid (RNA) synthesis are required during step 1, that DNA synthesis is not required, and that wall mucopeptide is not made. The reversion of well-primed protoplasts in the gelatin (step 2) proceeded undisturbed in thymine-starved cells with chromosomes arrested at the terminus. It was scarcely slowed by chloramphenicol in the gelatin but was delayed about 3 hr by both puromycin and actinomycin D. Escape from inhibition occurred while the inhibitors were still actively blocking growth. Penicillin and cycloserine inhibited and lysozyme reversed reversion. Momentary melting of the gelatin delayed reversion. It is concluded that mucopeptide synthesis occurs in step 2, that concomitant RNA, DNA, or protein synthesis is not essential, but that physical immobilization of excreted cell products at the protoplast surface is necessary early in step 2. Newly reverted cells were misshapen and osmotically sensitive. Processes which confer osmotic stability after reversion (step 3) did not occur in the presence of chloramphenicol or actinomycin D. 相似文献
12.
J M Sanger J S Dome B Mittal A V Somlyo J W Sanger 《Cell motility and the cytoskeleton》1989,13(4):301-319
The dynamic changes of the endoplasmic reticulum (ER) in interphase and mitotic cells was detected by the vital fluorescent dye 3,3'-dihexyloxacarbocyanine iodide. Two types of arrays characterize the continuous ER system in the non-muscle PtK2 cell: 1) a lacy network of irregular polygons and 2) long strands of ER that are found aligned along stress fibers. In cross-striated myotubes there was a periodic localization of fluorescence over each I-band corresponding to the positions of the terminal cisternae of the sarcoplasmic reticulum (SR). In contrast to the arrangement in muscle cells, the alignment of the long strands of ER alon stress fibers showed no strict periodicity that could be correlated with the sarcomeric units of the stress fibers. The ER and SR arrays seen in living cells were also detected in fixed cells stained with antibodies directed against proteins of the endoplasmic reticulum and sarcoplasmic reticulum, respectively. Observations of vitally stained PtK2 cells at 1 to 2 minute intervals using low light level video cameras and image processing techniques enabled us to see the polygonal ER units form and undergo changes in their shapes. During cell division, the ER, rhodamine 123-stained mitochondria, and phagocytosed fluorescent beads were excluded from the mitotic spindle while soluble proteins were not. No obvious concentration or alignment of membranes could be found associated with the contractile proteins in the cleavage furrow. After completion of cell division there was a redeployment of the ER network in each daughter cell. 相似文献
13.
Haeffner EW Wittmann U Kiesewetter L Zimmermann HP Stöhr M Spiess E 《Cell biology international》2007,31(8):790-797
We have introduced a heterologous coculture model between Ehrlich ascites tumor (EAT) and baby hamster kidney cells (PtK2), and we have studied the influence of PtK2 cells and their newly synthesized cholesterol on uptake and tumor cell proliferation. PtK2 cells produce about five times more cholesterol as compared to EAT cells, and they support tumor cell growth in coculture experiments. This growth stimulation is reduced by 80% when EAT cells are cultured in PtK2 cell-derived medium in the presence of a monoclonal anti-low-density lipoprotein receptor (anti-LDL(r)) antibody. Freshly synthesized cholesterol by PtK2 cells is taken up by EAT cells in a time-dependent manner amounting to a threefold increase after 24 h. Alternatively, cholesterol transfer to EAT cells is decreased between 28% and 35%, when PtK2 cell cholesterol synthesis is inhibited in the presence of mevinolin, the specific inhibitor of the hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) reductase. Furthermore, lower levels of EAT cell LDL receptor induced by antisense technology reduces cholesterol uptake and cell proliferation. These data demonstrate a metabolic interaction between normal and tumor cells mediated via the exchange of cholesterol, an important membrane constituent. 相似文献
14.
Alteration of the distribution of intermediate filaments in PtK1 cells by acrylamide. II: Effect on the organization of cytoplasmic organelles 总被引:1,自引:0,他引:1
B S Eckert 《Cell motility and the cytoskeleton》1986,6(1):15-24
The distribution and motility of cytoplasmic particles was examined in PtK1 cells in which intermediate filament networks had been disrupted by acrylamide. In these cells, particles (mitochondria and vesicles) accumulated near the cell center although saltatory movements continued. This left a broad sheet of agranular cytoplasm at the periphery of the cell. Particles were capable of movement into this sheet. Intermediate filaments were absent in the peripheral cytoplasm although microtubules remained in a normal configuration. Particles apparently move along the microtubules. These results indicate that particle movement along microtubules is not dependent upon the normal configuration of intermediate filaments. It is suggested that intermediate filaments are necessary for normal organelle distribution and serve as a matrix with which particles can associate to maintain position. 相似文献
15.
A microtubule-associated protein antigen unique to mitotic spindle microtubules in PtK1 cells 总被引:21,自引:17,他引:4
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Microtubule-associated proteins (MAPs) that copurify with tubulin through multiple cycles of in vitro assembly have been implicated as regulatory factors and effectors in the in vivo activity of microtubules. As an approach to the analysis of the functions of these molecules, a collection of lymphocyte hybridoma monoclonal antibodies has been generated using MAPs from HeLa cell microtubule protein as antigen. Two of the hybridoma clones secrete IgGs that bind to distinct sites on what appears to be a 200,000-dalton polypeptide. Both immunoglobulin preparations stain interphase and mitotic apparatus microtubules in cultured human cells. One of the clones (N-3B4.3.10) secretes antibody that reacts only with cells of human origin, while antibody from the other hybridoma (N-2B5.11.2) cross-reacts with BSC and PtK1 cells, but not with 3T3 cells. In PtK1 cells the N-2B5 antigen is associated with the microtubules of the mitotic apparatus, but there is no staining of the interphase microtubule array; rather, the antibody stains an ill-defined juxtanuclear structure. Further, neither antibody stains vinblastine crystals in either human or marsupial cells at any stage of the cell cycle. N-2B5 antibody microinjected into living PtK1 cells binds to the mitotic spindle, but does not cause a rapid dissolution of either mitotic or interphase microtubule structures. When injected before the onset of anaphase, however, the N-2B5 antibody inhibits proper chromosome partition in mitotic PtK1 cells. N-2B5 antibody injected into interphase cells causes a redistribution of MAP antigen onto the microtubule network. 相似文献
16.
One of the two nucleoli of tetraploid PtK2 WA cells in early prophase was irradiated with an ultraviolet (UV) laser microbeam. The daughter cells that maintained the nucleolar deficiency were isolated and cloned. Five nucleolar deficient sublines of PtK2 WA were established, thus providing an experimental system to study the ribosomal gene-nucleolar organizer complex. 相似文献
17.
Scatter factor affects major changes in the cytoskeletal organization of epithelial cells. 总被引:3,自引:0,他引:3
The effects of scatter factor on the cytoskeleton of MDCK and PtK2 cells are described. During the first 6 h after the addition of scatter factor, MDCK cells were found to increase their projected areas twofold, as well as the number and size of their F-actin stress fibers. In contrast PtK2 cells showed no change in their projected areas or in their stress fiber content. However, when both MDCK and PtK2 cells began to separate and scatter after approximately 6 h, the size and number of stress fibers was found to decrease considerably. Unscattered PtK2 cells and cells treated with scatter factor which had yet to scatter showed focal contacts present over the whole ventral surface, as judged by staining for both vinculin and talin. After treated cells separated, both vinculin and talin staining were mainly present in focal contacts on the ventral surfaces of the cell bodies and the distal ends of the processes. However, the cell processes showed few focal contacts along their lengths. The distribution of microtubules and vimentin and keratin intermediate filaments also did not change significantly until scattering had occurred. After cell separation, the processes were always packed with microtubules which were often, but not always, rich in detyrosinated alpha-tubulin and often, but not always, packed with intermediate filaments. All these changes in cytoskeletal organization are consistent with the adoption of a much more motile phenotype. The changes found are compared with those brought about by transformation. 相似文献
18.
Prosomes are small ribonucleoprotein (RNP) particles of unique morphology in the electron microscope but of variable protein and RNA composition, depending on the differentiation state of the cells studied. They were initially observed as subcomplexes of untranslated mRNP. In previous studies, we found that prosomes are associated to the intermediate filaments (IF) of cytokeratin type in HeLa and PtK1 cells. Here we have studied in detail the association of prosomal antigens with the IF networks in PtK1 cells. Contrary to our earlier conclusions, in these cells the vimentin fibers also carry prosomes which, thus, distribute in between the two types of networks. During the selective collapse of the IF induced by acrylamide, and upon recovery after the withdrawal of the drug, no dissociation of the prosome and IF networks of cytokeratin- and vimentin-type could be observed. These data show that even in a dynamic situation, prosome and IF antigens do not dissociate, indicating strongly that they are located on one and the same structure. Furthermore, the differential distribution of specific prosomal antigens between both types of intermediate filament networks indicates that prosomes do not ubiquitously populate the intermediate filaments but occupy subnetworks of either vimentin or cytokeratin type. 相似文献
19.
Norihiko Misawa Tomoyuki Okamoto Katsumi Nakamura Kumpei Kitamura Hideshi Yanase Kenzo Tonomura 《Bioscience, biotechnology, and biochemistry》2013,77(12):3201-3203
The culture conditions for Rhodococcus sp. N-774 cells showing high nitrile hydratase activity and the reaction conditions for acrylamide production by the resting cells were optimized. Thiamine was essential for the growth of the strain. Yeast extract and Fe2 + or Fe3 + remarkably promoted the formation of nitrile hydratase of the cells. The reaction proceeded optimally at temperatures below 30°C. Incubation for 1 hr at above 40°C resulted in inactivation of the enzyme. Through reaction at a temperature as low as 0°C, the inhibition and inactivation of the enzyme activity by the substrate, acrylonitrile, and the product, acrylamide, were remarkably reduced, and higher accumulation of acrylamide could be attained. Under the optimal conditions, a more than 20% (w/v) acrylamide solution was obtained with a conversion yield of nearly 100%. Thus, the aqueous acrylamide solution obtained showed a high enough quality for use for the commercial preparation of polyacrylamide. 相似文献
20.
PtK1 cells contain a nondiffusible, dominant factor that makes the Golgi apparatus resistant to brefeldin A 总被引:14,自引:5,他引:9
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Brefeldin A (BFA) was shown in earlier studies of numerous cell types to inhibit secretion, induce enzymes of the Golgi stacks to redistribute into the ER, and to cause the Golgi cisternae to disappear. Here, we demonstrate that the PtK1 line of rat kangaroo kidney cells is resistant to BFA. The drug did not disrupt the morphology of the Golgi complex in PtK1 cells, as judged by immunofluorescence using antibodies to 58- (58K) and 110-kD (beta-COP) Golgi proteins, and by fluorescence microscopy of live cells labeled with C6-NBD-ceramide. In addition, BFA did not inhibit protein secretion, not alter the kinetics or extent of glycosylation of the vesicular stomatitis virus (VSV) glycoprotein (G-protein) in VSV-infected PtK1 cells. To explore the mechanism of resistance to BFA, PtK1 cells were fused with BFA-sensitive CV-1 cells that had been infected with a recombinant SV-40 strain containing the gene for VSV G-protein and, at various times following fusion, the cultures were exposed to BFA. Shortly after cell fusion, heterokaryons contained one Golgi complex associated with each nucleus. Golgi membranes derived from CV-1 cells were sensitive to BFA, whereas those of PtK1 origin were BFA resistant. A few hours after fusion, most heterokaryons contained a single, large Golgi apparatus that was resistant to BFA and contained CV-1 galactosyltransferase. In unfused cells that had been perforated using nitrocellulose filters, retention of beta-COP on the Golgi was optimal in the presence of cytosol, ATP, and GTP. In perforated cell models of the BFA-sensitive MA104 line, BFA caused beta-COP to be released from the Golgi complex in the presence of nucleotides, and either MA104 or PtK1 cytosol. In contrast, when perforated PtK1 cells were incubated with BFA, nucleotides, and cytosol from either cell type, beta-COP remained bound to the Golgi complex. We conclude that PtK1 cells contain a nondiffusible factor, which is located on or very close to the Golgi complex, and confers a dominant resistance to BFA. It is possible that this factor is homologous to the target of BFA in cells that are sensitive to the drug. 相似文献