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1.
The continuous fermentation of 1,3-propanediol from glycerol by Clostridium butyricum was subjected to cell recycling by filtration using hollow-fibre modules made from polysulphone. The performance of the culture
system was checked at a retention ratio (dilution rate/bleed rate) of 5, dilution rates between 0.2 h−1 and 1.0 h−1 and glycerol input concentrations of 32 g l−1 and 56 g l−1. The near-to-optimum propanediol concentration of 26.5 g l−1 (for 56 g l−1 glycerol) was maintained up to a dilution rate of 0.5 h−1 and then decreased while the propanediol productivity was highest at 0.7 h−1. The productivity could be increased by a factor of four in comparison to the continuous culture without cell recycling.
By application of the model of Zeng and Deckwer [(1995) Biotechnol Prog 11: 71–79] for cultures under substrate excess, it
was shown that the limitations resulted exclusively from product inhibition and detrimental influences from the cell recycling
system, such as shear stress, were not involved.
Received: 20 October 1997 / Received revision: 12 December 1997 / Accepted: 14 December 1997 相似文献
2.
Demonstration of two distinct transferrin receptor recycling pathways and transferrin-independent receptor internalization in K562 cells 总被引:6,自引:0,他引:6
The endocytosis and recycling of the human transferrin receptor were evaluated by several experimental modalities in K562 cells perturbed with 10(-5) M monensin. The work presented is an extension of a previous study demonstrating both complete inhibition of release of internalized human transferrin and a 50% reduction in the number of cell surface transferrin binding sites in K562 cells treated with monensin (Stein, B. S., Bensch, K. G., and Sussman, H. H. (1984) J. Biol. Chem. 259, 14762-14772). The data directly reveal the existence of two distinct transferrin receptor recycling pathways. One pathway is monensin-sensitive and is felt to represent recycling of transferrin receptors through the Golgi apparatus, and the other pathway is monensin-resistant and most likely represents non-Golgi-mediated transferrin receptor recycling. A transferrin-free K562 cell culture system was developed and used to demonstrate that cell surface transferrin receptors can be endocytosed without antecedent ligand binding, indicating that there are factors other than transferrin binding which regulate receptor internalization. Evidence is presented suggesting that two transferrin receptor recycling pathways are also operant in K562 cells under ligand-free conditions, signifying that trafficking of receptor into either recycling pathway is not highly ligand-dependent. 相似文献
3.
Lawrence E. Shapiro Neil Wagner 《In vitro cellular & developmental biology. Plant》1989,25(7):650-654
Summary We have previously reported that Reuber H-35 rat hepatoma cells secrete an autocrine growth-stimulating activity in serum-free
culture. To characterize this activity, conditioned serum-free medium from dense H-35 donor cultures was collected in the
absence and presence of [35S]methionine. A 1∶4 dilution of conditioned medium into fresh serum-free medium resulted in an increase in mean H-35 cell
numbers per assay dish from 1.59±0.12×105 to 3.35±0.34×105 after 44 h of incubation. Control, unconditioned medium, resulted in significantly (P<0.05) less growth (2.14±0.41×105 cells per dish). Trypsin digestion eliminated the growth-promoting effect of conditioned medium but had no effect on unconditioned
medium. Dialysis did not diminish the growth-promoting activity of conditioned medium. The immunoprecipitate of [35S]methionine-containing conditioned medium with antisera against rat serum transferrin contained a dominant radioactive doublet
of molecular weight equal to purified rat serum transferrin. A rat transferrin radioimmunoassay was devised and used to quantitate
that 29.1±1.2 ng of transferrin was secreted per 106 cells per hour in serum-free culture. Addition of antitransferrin antibody resulted in a significant (P<0.025) decrease in H-35 cell growth after 48 h. Thus, a portion of the autocrine growth-promoting activity secreted by H-35
cells into serum-free culture is due to transferrin.
This work was funded by a feasibility grant from the American Diabetes Association, as well as by grants CA 24604-09 and CA
16463-14 from The National Institutes of Health, Bethesda, MD. 相似文献
4.
Long-term multiplication of the Chinese hamster ovary (CHO) cell line in a serum-free medium 总被引:2,自引:0,他引:2
Francois Gasser Philippe Mulsant Michel Gillois 《In vitro cellular & developmental biology. Plant》1985,21(10):588-592
Summary A new synthetic medium (referred to as GC3) that supports the growth of the Chinese hamster ovary cell line has been developed. It is composed of a 1∶1 mixture of Ham's
F12 and modified Eagle's minimum essential (MEM.S) mediums supplemented with transferrin (10 μg/ml), insulin (80 mU/ml), and
selenium (1×10−7
M). Other more simple supplementations of our basal medium MEM.S/F12 (transferrin+insulin, transferrin+selenium, ferrous iron+selenium)
also give good cell growth responses. Fibronectin or serum pretreatment is not needed for cellular attachment and spreading.
Our culture system is characterized by a continuous serum-free cultivation (more than 200 doublings), a clonal growth, a high
density proliferation, and a rapid growth rate near that of cells in serum-supplemented medium. 相似文献
5.
Carlozzi P 《Applied microbiology and biotechnology》2000,54(1):14-22
Two tubular undulating row photobioreactors (TURPs) with a very high illuminated surface/volume ratio (400 m−1) were designed and constructed for the growth of photosynthetic micro-organisms. Experiments were conducted under outdoor
conditions; and Arthrospira recycling was performed with airlifts (one for each row). The rows in each reactor faced east-west and consisted of a flexible
polyvinyl chloride pipe (22 m long, 0.01 m bore) arranged in a sinusoidal shape. We studied the hydraulic performance of the
sine-shaped photobioreactor rows during culture recycling in the TURPs at a very high Reynolds number (4200), when Arthrospira showed Newtonian fluid behavior. The sinusoidal pipe arrangement imposed a sine waveform on the culture, which led to better
light utilization. During summer, a volumetric productivity of 2.2 g l−1 day−1 was reached in the TURP-5r (5 rows m−2), whereas an area productivity of 35 g m−2 day−1 was obtained in the TURP-10r (10 rows m−2). This was due to more light being available in the TURP-5r, because its rows were more spaced out and the photic ratio (R
f) was low (3.0). In the TURP-10r, the closer rows caused a dilution of the sunlight, but gave a better light distribution
inside the Arthrospira culture and improved the light utilization. This was attributed to the high R
f (6.0) of this reactor.
Received: 8 October 1999 / Received revision: 20 January 2000 / Accepted: 23 January 2000 相似文献
6.
Zohra Chikh Nguyêt-Thanh Ha-Duong Geneviève Miquel Jean-Michel El Hage Chahine 《Journal of biological inorganic chemistry》2007,12(1):90-100
The kinetics and thermodynamics of Ga(III) exchange between gallium mononitrilotriacetate and human serum transferrin as well
as those of the interaction between gallium-loaded transferrin and the transferrin receptor 1 were investigated in neutral
media. Gallium is exchanged between the chelate and the C-site of human serum apotransferrin in interaction with bicarbonate
in about 50 s to yield an intermediate complex with an equilibrium constant K
1 = (3.9 ± 1.2) × 10−2, a direct second-order rate constant k
1 = 425 ± 50 M−1 s−1 and a reverse second-order rate constant k
−1 = (1.1 ± 3) × 104 M−1 s−1. The intermediate complex loses a single proton with proton dissociation constant K
1a = 80 ± 40 nM to yield a first kinetic product. This product then undergoes a modification in its conformation which lasts
about 500 s to produce a second kinetic intermediate, which in turn undergoes a final extremely slow (several hours) modification
in its conformation to yield the gallium-saturated transferrin in its final state. The mechanism of gallium uptake differs
from that of iron and does not involve the same transitions in conformation reported during iron uptake. The interaction of
gallium-loaded transferrin with the transferrin receptor occurs in a single very fast kinetic step with a dissociation constant
K
d = 1.10 ± 0.12 μM and a second-order rate constant k
d = (1.15 ± 0.3) × 1010 M−1 s−1. This mechanism is different from that observed with the ferric holotransferrin and suggests that the interaction between
the receptor and gallium-loaded transferrin probably takes place on the helical domain of the receptor which is specific for
the C-site of transferrin and HFE. The relevance of gallium incorporation by the transferrin receptor-mediated iron-acquisition
pathway is discussed. 相似文献
7.
Adam H. Balen Jovita Er Brian Rafferty Matthew Rose 《In vitro cellular & developmental biology. Animal》1995,31(4):316-322
Summary We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible
bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure
the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling
of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration
of 0.4 × 106 cells · ml−1 · well−1 in serum-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture, supplemented with insulin and transferrin) in
Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further
48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10−8
M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All
samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize
plate effects. Random number tables were used to achieve this randomization. 相似文献
8.
Jingsheng Gu 《Experimental cell research》2010,316(12):1946-1588
Mechanisms for receptor-mediated anthrax toxin internalization and delivery to the cytosol are well understood. However, far less is known about the fate followed by anthrax toxin receptors prior and after cell exposure to the toxin. We report that Anthrax Toxin Receptor 1/Tumor Endothelial Marker 8 (TEM8) localized at steady state in Rab11a-positive and transferrin receptor-containing recycling endosomes. TEM8 followed a slow constitutive recycling route of ∼ 30 min as determined by pulsed surface biotinylation and chase experiments. A Rab11a dominant negative mutant and Myosin Vb tail expression impaired TEM8 recycling by sequestering TEM8 in intracellular compartments. Sequestration of TEM8 in intracellular compartments with monensin coincided with increased TEM8 association with a multi-protein complex isolated with antibodies against transferrin receptor. Addition of the cell-binding component of anthrax toxin, Protective Antigen, reduced TEM8 half-life from 7 to 3 hours, without preventing receptor recycling. Pharmacological and molecular perturbation of recycling endosome function using monensin, dominant negative Rab11a, or myosin Vb tail, reduced PA binding efficiency and TEM8-dependent cell spreading on PA-coated surfaces without affecting toxin delivery to the cytosol. These results indicate that the intracellular fate of TEM8 differentially affect its cell adhesion and cell intoxication functions. 相似文献
9.
Ellis D. Avner William E. Sweeney Jr. Nicholas P. Piesco Demetrius Ellis 《In vitro cellular & developmental biology. Plant》1985,21(5):297-304
Summary In order to define humoral growth factors which may regulate mammalian renal development, the growth requirements of fetal
metanephric organogenesis were studied in serum-free murine organ culture. Metanephric growth, determined by cell proliferation
and protein content, and metanephric differentiation, determined morphometrically as epithelial glomerular formation, were
compared and contrasted following 144 hours of organ culture incubation in basal medium, basal medium supplemented with 10%
fetal bovine serum, and basal medium supplemented with various combinations of growth factors. The basal medium was composed
of equal volumes of Dulbecco's modified Eagle's medium and Hams' F-12 medium. Five humoral growth factors were studied in
the following concentrations: selenium, 6.8×10−9 M; insulin, 8.3×10−7 M; triiodothyronine, 2×10−9 M; transferrin, 6.2×10−8 M; and prostaglandin E1, 7.1×10−8 M. Results showed that transferrin and prostaglandin E1 were necessary for optimal growth in the system and that prostaglandin E1 was necessary for maximal metanephric differentiation. Such data provide guidelines for the creation of serum-free medium
for future fetal renal cell and tissue culture systems, and provide insight into the factors which may regulate normal and
abnormal renal embryogenesis and the reparative processes of renal hyperplasia and hypertrophy which follow renal injury.
A preliminary report of this work was presented at the Ninth International Congress of Nephrology, Los Angeles, June 1984.
These studies were supported in part by Basil O'Connor Starter Research Grant 5-349 from the March of Dimes Birth Defects
Foundation and New Investigator Research Award I-R23-AM34891-01 from the National Institute of Arthritis, Diabetes, and Digestive
and Kidney Diseases of the National Institutes of Health (Both to Dr. Avner).
Editor's Statement The determination of effects of extracellular components on the introduction and maintenance of differentiation
is an area for which serum-free culture techniques are particularly suited. The approaches described in this report utilize
morphometric techniques to quantitate differentiation in serum-free fetal organ culture in addition to standard methodologies
for assessing growth. The purely epithelial nature of the cultures used in these studies also provides some interesting advantages
in the design of experiments aimed, at determining the importance of cell-cell interactions at various stages in the differentiative
process. David W. Barnes 相似文献
10.
Xylose-to-xylitol conversion was investigated in a bench-scale bioreactor using Candida guilliermondii cells entrapped within polyvinyl alcohol-hydrogel beads in a system operated in repeated-batch mode with cell recycling.
Yeast-viable cells were immobilized in the support using the freezing–thawing method. Bioconversion assays were performed
in a stirred tank reactor operated at 400-rpm agitation speed, 30°C temperature, and 1.04-vvm air flow rate. The system was
explored during six successive cycles, and a small decrease in the conversion performance in the fifth cycle was observed,
but the biocatalytic activity of the microorganism was recovered in the sixth cycle after washing the particles. During the
process, the hydrogel beads maintained their shape and size without appreciable deterioration. Xylitol production, yield factor,
and volumetric productivity increased with progressive recycling of cells and achieved their maximum values (P
F
= 39.7 g l−1; Y
P/S
= 0.77 g g−1; Q
P
= 0.53 g l−1 h−1, respectively) after the third cell recycling, probably because of cells’ adaptation to the medium. 相似文献
11.
Effect of prolactin on the testicular luteinizing hormone binding was studied in a serum-free culture system. By the collagenase
digestion of decapsulated testes taken out from 25-day-old rats, Leydig cells were isolated and cultured for 7 days in DME/F12
(1:1) medium supplemented with insulin, transferrin, epidermal growth factor, and gentamicin. The cultured cells exhibited
the 3β-hydroxysteroid dehydrogenase activity. Hill plots constructed from the data of competition experiment showed that the
dissociation constant (Kd) was 0.33 × 10–10M. The Kd value was approximately the same as the known value for the rat testicular homogenates. When the Leydig cells were cultured
with ovine prolactin for the last 3 days of 7-day culture period, the binding of luteinizing hormone increased to 1.7-fold
ofthat in the control group. From these results it is concluded that prolactin acts to up-regulate the binding of luteinizing
hormone to rat testicular Leydig cells in serum-free culture 相似文献
12.
Study of the dynamics of sertoli cell secretions in a new superfusion,two-compartment culture system
Andrzej Janecki Andrzej Jakubowiak Anna Steinberger 《In vitro cellular & developmental biology. Plant》1987,23(7):492-500
Summary A new superfusion, two-compaatment culture system recently developed in our laboratory was used to investigate the dynamic
changes in bidirectional secretion of transferrin, (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured
for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate
and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight
junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion
patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10−6
M; follicle stimulating hormone, 0.1 μg/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to
3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers
and for the study of bidirectional secretions under more “physiologic” conditions than those provided by static cultures.
This work was supported in part by grant HD 17802 (AS) from the National Institutes of Health, Bethesda, MD. 相似文献
13.
Culture of human hepatocytes from small surgical liver biopsies. Biochemical characterization and comparison with in vivo 总被引:4,自引:0,他引:4
M. José Gómez-Lechón Pilar López Teresa Donato Angel Montoya Amparo Larrauri Patricia Giménez Ramón Trullenque Ricardo Fabra José V. Castell 《In vitro cellular & developmental biology. Plant》1990,26(1):67-74
Summary High yields of human hepatocytes (up to 23×106 viable cells/g) were obtained from small surgical liver biopsies (1 to 3 g) by a two-step collagenase microperfusion method.
Cell viability was about 95%, attachment efficiency of hepatocytes seeded on fibronectin-coated plates was 80% within 1 h
after plating, and cells survived for about 2 wk in serum-free Ham’s F12 containing 0.2% bovine serum albumin, 10−8
M insulin, and 10−8
M dexamethasone. To evaluate the metabolism of human hepatocytes in serum-free conditions, we measured their most characteristic
biochemical functions and compared them to those reported for human liver. After 24 h in culture, glycogen content was 1250±177
nmol glucose/mg cell protein and remained stable for several days. Gluconeogenesis from lactate in hormone-free media was
(3.50±0.17 nmol glucose·mg−1·min−1) similar to that reported for human liver. Insulin at 10−8
M activated glycolysis (×1.40) and glycogenesis (×1.34), and glucagon at 10−9
M stimulated gluconeogenesis (×1.35) and glycogenolysis (×2.18). Human hepatocytes synthesized albumin, transferrin, fibrinogen,
α1-antitrypsin, α1-antichymotrypsin, α1-acid glycoprotein, haptoglobin, α2-macroglobulin, and plasma fibronectin and excreted them to the culture medium. Maximum protein synthesis was stimulated by
10−9
M dexamethasone. Basal urea synthesis oscillated between 2.5 and 3.5 nmol·mg−1 cell protein·min−1, about 5 times the value estimated for human liver. Cytochrome P-450 decreased in culture but it was still 20% of freshly
isolated hepatocytes by Day 5 in culture. In addition, ethoxycumarin-O-deethylase and aryl hydrocarbon hydroxylase could be
induced in vitro by treatment with methyl cholanthrene. Glutathione levels were similar to those reported for human liver
(35 nmol·mg−1).
The results of our work show that adult human hepatocytes obtained from small surgical biopsies and cultured in chemically
defined conditions express their most important metabolic functions to an extent that is similar to that reported for adult
human liver. 相似文献
14.
B. C. Lim E. H. Morgan 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1985,155(2):201-210
Summary The mechanism of iron uptake by avian erythroid cells was investigated using cells from 7 and 15-day chicken embryos, and chicken serum transferrin and conalbumin (ovotransferrin) labelled with125I and59Fe. Endocytosis of the protein was determined by incubation of the cells with Pronase at 4°C to distinguish internalized from surface-bound protein.Iron was taken up by the cells by receptor-mediated endocytosis of transferrin or conalbumin. The receptors had the same affinity for serum transferrin and conalbumin. Endocytosis of diferric transferrin and conalbumin and exocytosis of apo-protein occurred at the same rates, indicating that iron donation to the cells occurred during the process of intracellular cycling of the protein. The recycling time was approximately 4 min. The rate of endocytosis of diferric protein varied with incubation temperature and at each temperature the rate of endocytosis was sufficient to account for the iron accumulated by the cells. These results and experiments with a variety of inhibitors confirmed the role of endocytosis in iron uptake.The mean cell volumes, receptor numbers and iron uptake rates of 7-day embryo cells were approximately twice those of 15-day embryo cells but the protein recycling times were approximately the same. Hence, the level of transferrin receptors is probably the main determinant of the rate of iron uptake during development of chicken erythroid cells.Transferrins from a variety of mammalian species were unable to donate iron to the chicken cells, but toad (Bufo marinus) transferrin could do so at a slow rate. The mechanism of iron uptake by developing chicken erythroid cells appears to be similar to that described for mammalian cells, although receptor numbers and iron uptake rates are lower than those reported for mammalian cells at a similar stage of development.Abbreviations
BSS
Hanks balanced salt solution
-
PBS
phosphate buffered saline
-
MCV
mean corpuscular volume
-
CCCP
carbonyl cyanide-M-chlorophenyl hydrazone 相似文献
15.
Transferrin receptor 2 (TfR2), a homologue of the classical transferrin receptor 1 (TfR1), is found in two isoforms, α and β. Like TfR1, TfR2α is a type II membrane protein, but the β form lacks transmembrane portions and therefore is likely to be an intracellular protein. To investigate the functional properties of TfR2α, we expressed the protein with FLAG tagging in transferrin-receptor-deficient Chinese hamster ovary cells. The association constant for the binding of diferric transferrin (Tf) to TfR2α is 5.6 × 106 M− 1, which is about 50 times lower than that for the binding of Tf to TfR1, with correspondingly reduced rates of iron uptake. Evidence for Tf internalization and recycling via TfR2α without degradation, as in the TfR1 pathway, was also found. The interaction of TfR2α with Tf was further investigated using atomic force microscopy, a powerful tool used for investigating the interaction between a ligand and its receptor at the single-molecule level on the living cell surface. Dynamic force microscopy reveals a difference in the interactions of Tf with TfR2α and TfR1, with Tf-TfR1 unbinding characterized by two energy barriers, while only one is present for Tf-TfR2. We speculate that this difference may reflect Tf binding to TfR2α by a single lobe, whereas two lobes of Tf participate in binding to TfR1. The difference in the binding properties of Tf to TfR1 and TfR2α may help account for the different physiological roles of the two receptors. 相似文献
16.
Herman A. J. Schut Everard H. Hughes Snorri S. Thorgeirsson 《In vitro cellular & developmental biology. Plant》1981,17(4):275-283
Summary Radioimmunoassay was used to determine α-fetoprotein (AFP), albumin, and transferrin production (ng/105 cells/24 h) by two cell lines (7777 and 8994) derived from chemically induced rat hepatomas. α-Fetoprotein production was
high (2000 to 4400) in 7777, but was very low (0.2 to 0.4) in 8994. Albumin production varied from 0.4–0.8 (7777) to 14–26
(8994). Both lines produced substantial amounts of transferrin (180 to 240 by 7777 and 29 to 42 by 8994). Addition of dimethyl
sulfoxide (DMSO, 1 to 4%) or sodium butyrate (BA, 0.5 to 2.0 mM) to the medium inhibited growth in both lines, but 8994 was more sensitive to these agents than 7777. Dimethyl sulfoxide
treatment (2 to 4%) resulted in a dose-related decrease (<10% of control at 4% DMSO) in AFP, albumin, and transferrin production
by 7777, but in 8994, DMSO (1 to 2%) resulted in an increase, (up to sixfold) in albumin and transferrin production, without
affecting AFP production. By contrast, BA (2 to 4 mM) stimulated the production of all three proteins in both lines, most notably that of albumin (up to sixfold) by 7777 and
that of AFP (up to 20-fold) by 8994. It is concluded that both DMSO and BA can enhance the expression of differentiated functions
of the hepatoma cell, and that DMSO at the same time can suppress the expression of an oncofetal function. However, neither
DMSO nor BA is selective in its effects on specific genes (i.e., normal, adult vs. oncofetal genes), and it appears that their
effects may be the result of a more general phenomenon, the expression of which may be related to the stage of differentiation
of the cell. 相似文献
17.
Transferrin receptor mediates internalization of transferrin with bound ferric ions through the clathrin-dependent pathway. We found that binding of transferrin to the receptor induced rapid generation of cell surface ceramide which correlated with activation of acid, but not neutral, sphingomyelinase. At the onset of transferrin internalization both ceramide level and acid sphingomyelinase activity returned to their basic levels. Down-regulation of acid sphingomyelinase in cells with imipramine or silencing of the enzyme expression with siRNA stimulated transferrin internalization and inhibited its recycling. In these conditions colocalization of transferrin with clathrin was markedly reduced. Simultaneously, K+ depletion of cells which interfered with the assembly of clathrin-coated pits inhibited the uptake of transferrin much less efficiently than it did in control conditions. The down-regulation of acid sphingomyelinase activity led to the translocation of transferrin receptor to the raft fraction of the plasma membrane upon transferrin binding. The data suggest that lack of cell surface ceramide, generated in physiological conditions by acid sphingomyelinase during transferrin binding, enables internalization of transferrin/transferrin receptor complex by clathrin-independent pathway. 相似文献
18.
Baravalle G Schober D Huber M Bayer N Murphy RF Fuchs R 《Cell and tissue research》2005,320(1):99-113
The effects of bafilomycin, nocodazole, and reduced temperature on recycling and the lysosomal pathway have been investigated in various cultured cell lines and have been shown to vary dependent on the cell type examined. However, the way in which these treatments affect recycling and transport to lysosomes within the same cell line has not been analyzed. In the current study, we used fluorophore-labeled transferrin and dextran as typical markers for the recycling and the lysosomal pathways, respectively, to explore the morphology and the intravesicular pH of endocytic compartments in HeLa cells. The V-ATPase inhibitor bafilomycin selectively inhibited the transport of marker destined for lysosomal degradation in early endosomes, whereas the transport of transferrin to the perinuclear recycling compartment (PNRC) still occurred. The kinetics of transferrin acidification was found to be biphasic, indicative of fast and slow recycling pathways via early endosomes (pH 6.0) and PNRC (pH 5.6), respectively. Furthermore, the disruption of microtubules by nocodazole blocked the transport of transferrin to the PNRC in early endosomes and of lysosome-directed marker into endosomal carrier vesicles. In contrast, incubation at 20°C affected the lysosomal pathway by causing retention of internalized dextran in late endosomes and a delay in transferrin recycling. Taken together, these data clearly demonstrate, for the first time, that the transferrin recycling pathway and transport of endocytosed material to lysosomes are differentially affected by bafilomycin, nocodazole, and low temperature in HeLa cells. Consequently, these treatments can be applied to investigate whether internalized macromolecules such as viruses follow a recycling or degradative pathway.This work was supported by grants from the Austrian Science Fund P12967 and P17590 to R.F. 相似文献
19.
Plant regeneration from protoplast culture of Crocus cancellatus was investigated using regenerable embryogenic calli obtained from shoot meristem culture on LS (Linsmaier and Skoog, 1965)
medium containing 4 mg l−1 kinetin and 1 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D). Protoplasts were isolated directly from embryogenic calli. The best protoplast growth
was found on those embedded in Ca-alginate beads and cultured with nurse cells in MS (Murashige and Skoog, 1962) medium supplemented
with 2 mg l−1 kinetin, 1 mg l−1 2,4-D and 100 mg l−1 ascorbic acid at 25 °C in darkness. After 4–5 weeks of culture, microcalli appeared on the surface of the Ca-alginate beads,
but the protoplasts without immobilization in Ca-alginate beads showed very low cell division. Growth of the microcalli in
the medium with nurse cells was much better than in the medium without nurse cells. Transferring beads onto half strength
MS medium supplemented with 0.2 mg l−1 kinetin and 0.1 mg l−1 2,4-D, increased the growth of embryogenic calli. Somatic embryo development was observed either on half strength MS medium
growth regulator free or with 1 mg l−1 abscisic acid. Matured embryos germinated on half strength MS medium containing 25 mg l−1 of gibberelic acid. Plantlet formation was obtained on half strength MS medium containing 1 mg l−1 6-benzyladenine and 1 mg l−1 α-naphthaleneacetic acid at 20 °C in a 16/8 h light/dark cycle.
This revised version was published online in June 2006 with corrections to the Cover Date. 相似文献
20.
Growth and differentiation in cultured human thyroid cells: Effects of epidermal growth factor and thyrotropin 总被引:2,自引:0,他引:2
Janice E. Errick Katherine W. A. Ing Margaret C. Eggo Gerard N. Burrow 《In vitro cellular & developmental biology. Plant》1986,22(1):28-36
Summary Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and
to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture
conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of
insulin (10 μg/ml), transferrin (5 μg/ml), hydrocortisone (10 μg/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine
(10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present
at 10−9
M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system
provides a means to study the hormonal control of growth and differentiation in human thyroid cells.
This work was supported by grants from the Medical Research Council of Canada; the Department of Medicine, University of Toronto;
and the National Cancer Institute of Canada. J. E. E. is a C.H. Best Foundation and Department of Medicine postdoctoral fellow. 相似文献