共查询到20条相似文献,搜索用时 15 毫秒
1.
R Lüllmann-Rauch M Ziegenhagen 《Virchows Archiv. B, Cell pathology including molecular pathology》1991,60(2):99-104
Several di-cationic amphiphilic compounds are known to cause lysosomal accumulation of sulfated glycosaminoglycans (sGAG) in intact rats and in cultured rat fibroblasts. The purpose of the present investigation was to examine whether this drug side effect also occurs in bovine and human cells. Cultured fibroblasts from both species were exposed to tilorone (3 microM and 5 microM) for 72 h; lysosomal sGAG-storage was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The cytological alterations as well as the radiochemical results in both species were in good agreement with previous data from rat fibroblasts. The present findings indicate that the drug-induced lysosomal storage of sGAG is a species-independent phenomenon. Thus, cultured bovine and human fibroblasts are a suitable model for further studies concerning the as yet unknown molecular mechanisms underlying this adverse drug action. 相似文献
2.
Summary The purpose of the present investigation was to examine whether or not a di-cationic amphiphilic compound that is known (1) to be accumulated in lysosomes and (2) to form insoluble complexes with sulfated glycosaminoglycans (sGAG) in vitro, is able to interfere with the lysosomal degradation of sGAG, thus causing mucopolysaccharidosis (MPS) in cultured cells. Acridine Orange (AO) was chosen for this study since it is known to meet the above requirements. Cultured fibroblasts from rat cornea were exposed to AO (0.7 M to 30 M) for 72 h; tilorone served as reference compound. AO (1.75 M to 10 M) caused MPS in a concentration-dependent manner, higher concentrations were cytotoxic. MPS was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The sGAG-complexing properties of AO were demonstrated by using it as a fixative for the intralysosomal sGAG accumulated in tilorone-treated cells. The present findings give support to the working hypothesis that the MPS induced by di-cationic amphilphilic drugs is due to the formation of insoluble sGAG-drug complexes, with the result that the sGAG become resistant to lysosomal degradation. 相似文献
3.
Acridine Orange, a precipitant for sulfated glycosaminoglycans, causes mucopolysaccharidosis in cultured fibroblasts 总被引:1,自引:0,他引:1
The purpose of the present investigation was to examine whether or not a di-cationic amphiphilic compound that is known (1) to be accumulated in lysosomes and (2) to form insoluble complexes with sulfated glycosaminoglycans (sGAG) in vitro, is able to interfere with the lysosomal degradation of sGAG, thus causing mucopolysaccharidosis (MPS) in cultured cells. Acridine Orange (AO) was chosen for this study since it is known to meet the above requirements. Cultured fibroblasts from rat cornea were exposed to AO (0.7 microM to 30 microM) for 72 h; tilorone served as reference compound. AO (1.75 microM to 10 microM) caused MPS in a concentration-dependent manner, higher concentrations were cytotoxic. MPS was demonstrated by cytochemical staining with cuprolinic blue and by measuring the intracellular accumulation of [35S]-GAG. The sGAG-complexing properties of AO were demonstrated by using it as a fixative for the intralysosomal sGAG accumulated in tilorone-treated cells. The present findings give support to the working hypothesis that the MPS induced by di-cationic amphiphilic drugs is due to the formation of insoluble sGAG-drug complexes, with the result that the sGAG become resistant to lysosomal degradation. 相似文献
4.
Masayoshi Ono John W. Perry Takami Oka 《In vitro cellular & developmental biology. Plant》1981,17(2):121-128
Summary Cortisol was previously shown to elicit a concentration-dependent inhibition of α-lactalbumin accumulation in midpregnant
mouse mammary gland cultured in medium containing optimal concentrations of 5 μg/ml prolactin and insulin. In contrast, casein
accumulation under these conditions was progressively stimulated by addition of increasing amounts of cortisol (Ono, M.; Oka,
T. Cell 19: 473–480; 1980). In the present study we found that in the presence of a suboptimal concentration of 0.5 μg/ml
prolactin, 2.8×10−9
M to 2.8×10−7
M cortisol stimulated α-lactalbumin accumulation. Furthermore, higher concentrations of cortisol produced a smaller inhibition
of α-lactalbumin accumulation as compared to that obtained in cultures containing 5 μg/ml prolactin. The maximal increase
in α-lactalbumin accumulation attained in the presence of 1.4×10−8
M cortisol, 0.5 μg/ml prolactin, and insulin was comparable to that observed in culture containing 5 μg/ml prolactin and insulin.
Similar results were obtained in a cortisol concentration-response study of α-lactalbumin accumulation in cultures containing
a suboptimal concentration of 0.5 μg/ml human placental lactogen. Measurement of the rate of α-lactalbumin synthesis in cultured
tissue indicated that the opposing effects of low and high concentrations of cortisol on α-lactalbumin accumulation involved
an alteration in the rate of synthesis of the milk protein. In contrast to α-lactalbumin, the synthesis of casein was stimulated
in a concentration-dependent manner by addition of cortisol that acted synergistically with either 0.5 μg/ml or 5 μg/ml prolactin.
The maximal increases were obtained in the presence of 2.8×10−6
M cortisol. These results indicated that the action of cortisol on α-lactalbumin accumulation can be modulated by the concentration,
of prolactin and suggest that the interplay between cortisol and prolactin in regulation of α-lactalbumin synthesis may be
different from that involved in casein synthesis. 相似文献
5.
Xiaohong Liu Junwei Zeng Yandong Zhao Zhi Xiao Chuanqing Fang Huaizhen Ruan 《Neurochemical research》2010,35(5):804-810
In addition to the classic genomic effects, it is well known that glucocorticoids also have rapid, nongenomic effects on neurons.
In the present study, the effect of corticosterone (CORT) on ATP-induced Ca2+ mobilization in cultured dorsal root ganglion (DRG) neurons were detected with confocal laser scanning microscopy using fluo-4/AM
as a calcium fluorescent indicator that could monitor real-time alterations of intracellular calcium concentration ([Ca2+]i). ATP, an algesic agent, caused [Ca2+]i increase in DRG neurons by activation of P2X receptor. Pretreatment with CORT (1 nM–1 μM for 5 min) inhibited ATP-induced
[Ca2+]i increase in DRG neurons. The rapid inhibition of ATP-induced Ca2+ response by CORT was concentration-dependent, reversible and could be blocked by glucocorticoid receptor antagonist RU38486
(10 μM). Furthermore, the inhibitory effect of CORT was abolished by protein kinase A inhibitor H89 (10 μM), but was not influenced
by protein kinase C inhibitor Chelerythrine chloride (10 μM). On the other hand, membrane-impermeable bovine serum albumin-conjugated
corticosterone had no effect on ATP-induced [Ca2+]i transients. These observations suggest that a nongenomic pathways may be involved in the effect of CORT on ATP-induced
[Ca2+]i transients in cultured DRG neurons. 相似文献
6.
Indole-3-butyric acid at 25 μM with methyl jasmonate (MJ) at 100 μM in Panax ginseng synergistically stimulated both root growth and ginsenoside accumulation compared with 100 μM MJ alone. Productivity of ginsenoside
was 10 mg l−1 d−1 compared to 7.3 mg l−1 d−1 with MJ elicitation alone. 相似文献
7.
Łubgan D Jóźwiak Z Grabenbauer GG Distel LV 《Cellular & molecular biology letters》2009,14(1):113-127
Neoplastic cells frequently have an increased number of transferrin receptors. Coupling transferrin to an anti-neoplastic
drug has the potential to overcome multidrug resistance (MDR). The purpose of this study was to examine the distribution and
action of doxorubicin-transferrin conjugate (DOXTRF) in a leukaemia cell line (HL60), a multidrug-resistant leukaemia cell
line (HL60ADR) and a normal tissue cell line (human fibroblasts). The intracellular accumulation of DOX and DOX-TRF was monitored
by direct fluorescence. More DOX-TRF than free DOX was delivered to the tumour cells, and consecutively the levels of DNA
double-strand breaks and apoptosis increased even in the multidrug-resistant cell line. In the normal tissue cell line, DOX-TRF
did not accumulate, and therefore, the levels of DNA double-strand breaks and apoptosis did not increase. Cell viability was
determined using the MTT assay. The IC50 for DOX-TRF was lower than the IC50 value for the free drug in both leukaemia cell lines. The IC50 values for the HL60 cells were 0.08 μM for DOX and 0.02 μM for DOX-TRF. The IC50 values for HL60ADR cells were 7 μM for DOX and 0.035 μM for DOX-TRF. In conclusion, DOX-TRF was able to overcome MDR in the
leukaemia cell lines while having only a very limited effect on normal tissue cells. 相似文献
8.
Acid α-glucosidase (GAA) is a lysosomal enzyme that hydrolyzes glycogen to glucose. Deficiency of GAA causes Pompe disease. Mammalian GAA is synthesized as a precursor of ~ 110,000 Da that is N-glycosylated and targeted to the lysosome via the M6P receptors. In the lysosome, human GAA is sequentially processed by proteases to polypeptides of 76-, 19.4-, and 3.9-kDa that remain associated. Further cleavage between R200 and A204 inefficiently converts the 76-kDa polypeptide to the mature 70-kDa form with an additional 10.4-kDa polypeptide. GAA maturation increases its affinity for glycogen by 7-10 fold. In contrast to human GAA, processing of bovine and hamster GAA to the 70-kDa form is more rapid. A comparison of sequences surrounding the cleavage site revealed human GAA contains histidine at 201 while other species contain hydrophobic amino acids at position 201 in the otherwise conserved sequence. Recombinant human GAA (rhGAA) containing the H201L substitution was expressed in 293 T cells by transfection. Pulse chase experiments in 293 T cells expressing rhGAA with or without the H201L substitution revealed rapid processing of rhGAAH201L but not rhGAAWT to the 70-kDa form. Similarly, when GAA precursor was endocytosed by human Pompe fibroblasts rhGAAH201L but not rhGAAWT was rapidly converted to the 70-kDa mature GAA. These studies indicate that the amino acid at position 201 influences the rate of conversion of 76-kDa GAA to 70-kDa GAA. The GAA sequence rather than the lysosomal protease environment explains the predominance of the 76-kDa form in human tissues. 相似文献
9.
Davis GD Masilamoni JG Arul V Kumar MS Baraneedharan U Paul SF Sakthivelu IV Jesudason EP Jayakumar R 《Cell biology and toxicology》2009,25(4):331-340
During the course of cancer radiation treatment, normal skin invariably suffers from the cytotoxic effects of γ-radiation
and reactive oxygen species (ROS), which are generated from the interaction between radiation and the water molecules in cells.
The present study was designed to investigate the radioprotective role of α-lipoic acid (LA), an antioxidant on murine skin
fibroblasts exposed to a single dose of 2, 4, 6, or 8Gy γ-radiation. Irradiation of fibroblasts significantly increased ROS,
nitric oxide, and lipid peroxidation (P < 0.001); all of these factors substantially decreased with 100 μM LA treatment. Hydroxyl radical (OH⋅) production from 8Gy irradiated fibroblasts was measured directly by electron spin resonance using spin-trapping techniques.
LA was found to inhibit OH⋅ production at 100-μM concentrations. Dose-dependent depletion of antioxidants, such as catalase and glutathione reductase,
was observed in irradiated fibroblasts (P < 0.001), along with increased superoxide dismutase (P < 0.001). LA treatment restored antioxidant levels. Concentration of the pro-inflammatory cytokine IL-1β was significantly
reduced in irradiated fibroblasts when treated with LA. MTT and lactate dehydrogenase assays demonstrated that LA treatment
reduced cell injury and protected cells against irradiation-induced cytotoxicity. Thus, we conclude that results are encouraging
and need further experiments to demonstrate a possible benefit in cancer patients and the reduction of harmful effects of
radiation therapy. 相似文献
10.
Jian Wu Henk Schat Rifei Sun Maarten Koornneef Xiaowu Wang Mark G. M. Aarts 《Plant and Soil》2007,291(1-2):167-180
Brassica rapa L. is an important vegetable crop in eastern Asia. The objective of this study was to investigate the genetic variation in
leaf Zn, Fe and Mn accumulation, Zn toxicity tolerance and Zn efficiency in B. rapa. In total 188 accessions were screened for their Zn-related characteristics in hydroponic culture. In experiment 1, mineral
assays on 111 accessions grown under sufficient Zn supply (2 μM ZnSO4) revealed a variation range of 23.2–155.9 μg g−1 dry weight (d. wt.) for Zn, 60.3–350.1 μg g−1 d. wt. for Fe and 20.9–53.3 μg g−1 d. wt. for the Mn concentration in shoot. The investigation of tolerance to excessive Zn (800 μM ZnSO4) on 158 accessions, by using visual toxicity symptom parameters (TSPs), identified different levels of tolerance in B. rapa. In experiment 2, a selected sub-set of accessions from experiment 1 was characterized in more detail for their mineral accumulation
and tolerance to excessive Zn supply (100 μM and 300 μM ZnSO4). In this experiment Zn tolerance (ZT) determined by relative root or shoot dry biomass varied about 2-fold. The same six
accessions were also examined for Zn efficiency, determined as relative growth under 0 μM ZnSO4 compared to 2 μM ZnSO4. Zn efficiency varied 1.8-fold based on shoot dry biomass and 2.6-fold variation based on root dry biomass. Zn accumulation
was strongly correlated with Mn and Fe accumulation both under sufficient and deficient Zn supply. In conclusion, there is
substantial variation for Zn accumulation, Zn toxicity tolerance and Zn efficiency in Brassica rapa L., which would allow selective breeding for these traits. 相似文献
11.
Eun-A Kim Hoh-Gyu Hahn Key-Sun Kim Tae Ue Kim Soo Young Choi Sung-Woo Cho 《Cellular and molecular neurobiology》2010,30(5):807-815
We have screened new drugs with a view to developing effective drugs against glutamate-induced excitotoxicity. In the present
work, we show effects of a new drug, 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride against glutamate-induced excitotoxicity
in primary rat glial cultures. Pretreatment of glial cells with 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride for
2 h significantly protected glial cells against glutamate-induced excitotoxicity in a time- and dose-dependent manner with
an optimum concentration of 100 μM. The drug significantly reduced production of proinflammatory cytokines, tumor necrosis
factor-α, and interlukin-1β in glutamate-induced excitotoxicity. The drug also prevented glutamate-induced intracellular Ca2+ influx and reduced the subsequent overproduction of nitric oxide and reactive oxygen species. Furthermore, the drug preserved
the mitochondrial potential and inhibited the overproduction of cytochrome c. In addition, the drug effectively attenuated the protein level changes of β-catenin and glycogen synthase kinase-3β. These
results suggest that 2-cyclopropylimino-3-methyl-1,3-thiazoline hydrochloride effectively protected primary cultures of rat
glial cells against glutamate-induced excitotoxicity. 相似文献
12.
The accumulation of [3H]inositol by mechanically dissociated brain cells and cultured skin fibroblasts from fetal mice was examined. Uptake by both tissues was strongly dependent on temperature and the presence of sodium ions. Brain and fibroblast uptake also responded similarly to inhibition by inositol isomers and phloridzin. At lower concentrations of inositol, both tissues exhibited high-affinity uptake kinetics with apparent Km values near 30 M, similar to values observed previously in human fibroblasts and other cultured cells. The activity of brain high-affinity uptake was nearly an order of magnitude lower than that of fibroblasts, however, and was in part confounded by the presence of a low-affinity or simple diffusion system operating at inositol concentrations above 100M. Brain preparation from adult mice also showed evidence of high-affinity, Na+ dependent uptake, but its activity was significantly diminished relative to that of fetal brain preparations. Our results demonstrate that a high-affinity inositol transport system closely resembling that found in cultured cells is expressed in the developing mouse brain. 相似文献
13.
U. A. Jo H. N. Murthy E. J. Hahn K. Y. Paek 《In vitro cellular & developmental biology. Plant》2008,44(1):26-32
An efficient, simple micropropagation method was developed for Alocasia amazonica using corms in semisolid and liquid cultures. Explants were cultured onto Murashige and Skoog (MS) medium (Murashige and
Skoog, Physiol. Plant. 15:473–497, 1962) supplemented with different cytokinins (Benzyladenine [BA, 2.22–13.32 μM], kinetin [2.32–13.95 μM], Thidiazuron [TDZ, 0.45–4.54 μM])
and cytokinin in combination with auxins [naphthalene acetic acid (NAA, 0.54–5.37 μM)/indole acetic acid (IAA, 0.57–5.71 μM)/indole
butyric acid (IBA, 0.49–4.9 μM)]. All supplementary-induced shoot proliferation and the optimal results was on the medium
supplemented with 2.27 μM TDZ, which induced 5.1 shoots per explant. Among the different concentrations of sucrose (0–120 g
l−1) tested for shoot proliferation, 30 g l−1 was found suitable for corm cultures of Alocasia amazonica. The optimal shoot proliferation and biomass values were with the plantlets grown at 30 μmol m−2 s−1 photosynthetic photon flux (PPF) and 25°C. Liquid cultures found suitable for shoot proliferation and biomass accumulation
was compared to semisolid cultures. Comparative studies of bioreactor systems [continuous immersion (with or without net)
and temporary immersion in liquid media using ebb and flood] revealed that shoot multiplication and growth were greatest with
the raft bioreactor system. Plantlets (cormlets) from the bioreactor were hydroponically cultured for 30 days, and 100% of
plants were acclimatized successfully. The simple efficient method of production of plantlets (cormlets) is useful for large-scale
multiplication of this important ornamental plant.
An erratum to this article can be found at 相似文献
14.
Nicotinic acetylcholine receptors (nAChR) are diverse members of the ligand-gated ion channel superfamily of neurotransmitter
receptors and play critical roles in chemical signaling throughout the nervous system. Reports of effects of substance P (SP)
on nAChR function prompted us to investigate interactions between several tachykinins and human nAChR subtypes using clonal
cell lines as simple experimental models. Acute exposure to SP inhibits carbamylcholine- or nicotinestimulated function measured
using86Rb+ efflux assays of human ganglionic (α3β4) nAChR expressed in SH-SY5Y neuroblastoma cells (IC50∼2.3 μM) or of human muscle-type (α1β1γδ) nAChR expressed in TE671/RD clonal cells (IC50∼21 μM). SP also acutely blocks function of rat ganglionic nAChR expressed in PC12 pheochromocytoma cells (IC50∼2.1 μM). Neurokinin A and eledoisin inhibit function (extrapolated IC50 values between 60 and 160 μM) of human muscle-type or ganglionic nAChR, but neurokinin B does not, and neither human nAChR
is as sensitive as PC12 cell α3β4-nAChR to eledoisin or neurokinin A inhibition. At concentrations that produce blockade of
nAChR function, SP fails to affect binding of [3H]acetylcholine to human muscle-type or ganglionic nAChR. SP-mediated blockade of rat or human ganglionic nAChR function is
insurmountable by increasing agonist concentrations. Collectively, these results indicate that tachykinins act noncompetitively
to inhibit human nAChR function with potencies that vary across tachykinins and nAChR subtypes. They also indicate that tachykinin
actions at nAChR could further contribute to complex cross-talk between nicotinic cholinergic and tachykinin signals in regulation
of nervous system activity. 相似文献
15.
Dynamics of zinc uptake and accumulation in the hyperaccumulating and non-hyperaccumulating ecotypes of Sedum alfredii Hance 总被引:2,自引:0,他引:2
X. E. Yang T. Q Li X. X. Long Y. H. Xiong Z. L. He P. J. Stoffella 《Plant and Soil》2006,284(1-2):109-119
Sedum alfredii Hance has been identified as a Zn-hyperaccumulating plant species native to China. The characteristics of Zn uptake and accumulation
in the hyperaccumulating ecotype (HE) and non-hyperaccumulating ecotype (NHE) of S. alfredii were investigated under nutrient solution and soil culture conditions. The growth of HE was normal up to 1000 μM Zn in nutrient solution, and 1600 mg Zn kg−1 soil in a Zn-amended soil. Growth of the NHE was inhibited at Zn levels ≥250 μM in nutrient solution. Zinc concentrations in the leaves and stems increased with increasing Zn supply levels, peaking at
500 and 250 μM Zn in nutrient solution for the HE and the NHE, respectively, and then gradually decreased or leveled off with further increase
in solution Zn. Minimal increases in root Zn were noted at Zn levels up to 50 μM; root Zn sharply increased at higher Zn supply. The maximum Zn concentration in the shoots of the HE reached 20,000 and
29,000 mg kg−1 in the nutrient solution and soil experiments, respectively, approximately 20 times greater than those of the NHE. Root Zn
concentrations were higher in the NHE than in the HE when plants were grown at Zn levels ≥50 μM. The time-course of Zn uptake and accumulation exhibited a hyperbolic saturation curve: a rapid linear increase during the
first 6 days in the long-term and 60 min in the short-term studies; followed by a slower increase or leveling off with time.
More than 80% of Zn accumulated in the shoots of the HE at half time (day 16) of the long-term uptake in 500 μM Zn, and also at half time (120 min) of the short-term uptake in 10 μM 65Zn2+. These results indicate that Zn uptake and accumulation in the shoots of S. alfredii exhibited a down-regulation by internal Zn accumulated in roots or leaves under both nutrient solution and soil conditions.
An altered Zn transport system and increased metal sequestration capacity in the shoot tissues, especially in the stems, may
be the factors that allow increased Zn accumulation in the hyperaccumulating ecotype of S. alfredii.
Section Editor: F. J. Zhao 相似文献
16.
Ya Fatou Njie-Mbye Odelia Y. N. Bongmba Chinwe C. Onyema Abhishek Chitnis Madhura Kulkarni Catherine A. Opere Angela M. LeDay Sunny E. Ohia 《Neurochemical research》2010,35(3):487-494
Hydrogen sulfide (H2S) has been reported to exert pharmacological effects on neural and non-neural tissues from several mammalian species. In
the present study, we examined the role of the intracellular messenger, cyclic AMP in retinal response to H2S donors, sodium hydrosulfide (NaHS) and sodium sulfide (Na2S) in cows and pigs. Isolated bovine and porcine neural retinae were incubated in oxygenated Krebs buffer solution prior to
exposure to varying concentrations of NaHS, Na2S or the diterpene activator of adenylate cyclase, forskolin. After incubation at different time intervals, tissue homogenates
were prepared for cyclic AMP assay using a well established methodology. In isolated bovine and porcine retinae, the combination
of both phosphodiesterase inhibitor, IBMX (2 mM) and forskolin (10 μM) produced a synergistic increase (P < 0.001) in cyclic AMP concentrations over basal levels. NaHS (10 nM–100 μM) produced a time-dependent increase in cyclic
AMP concentrations over basal levels which reached a maximum at 20 min in both bovine and porcine retinae. At this time point,
both NaHS and Na2S (10 nM–100 μM) caused a significant (P < 0.05) dose-dependent increase in cyclic AMP levels in bovine and porcine retinae. For instance, NaHS (100 nM) elicited
a four-fold and three-fold increase in cyclic AMP concentrations in bovine and porcine retinae respectively whilst higher
concentrations of Na2S (100 μM) produced a much lesser effect in both species. In bovine and porcine retinae, the effects caused by forskolin (10 μM)
on cyclic AMP production were not potentiated by addition of low or high concentrations of both NaHS and Na2S. We conclude that H2S donors can increase cyclic AMP production in isolated neural retinae from cows and pigs. Bovine retina appears to be more
sensitive to the stimulatory effect of H2S donors on cyclic nucleotide production than its porcine counterpart indicating that species differences exist in the magnitude
of this response. Furthermore, effects produced by forskolin on cyclic AMP formation were not additive with those elicited
by H2S donors suggesting that these agents may share a common mechanism in their action on the adenylyl cyclase pathway. 相似文献
17.
Chandra P Lecluyse EL Brouwer KL 《In vitro cellular & developmental biology. Animal》2001,37(6):380-385
Summary This study was undertaken to examine the influence of time and volume of collagen overlay, type of media, and media additives
on taurocholate (TC) accumulation and biliary excretion in hepatocytes cultured in a collagen-sandwich configuration. Hepatocytes
were isolated from male Wistar rats by in situ perfusion with collagenase, seeded onto collagencoated 60-mm dishes, overlaid
with gelled collagen, and cultured for 4 d. Experiments to examine the influence of time and volume of collagen overlay were
conducted in Dulbecco's modified Eagle's medium (DMEM)+1.0μM dexamethasone (DEX)+5% fetal bovine serum (FBS). Hepatocytes were overlaid at 0 h with 0.1 or 0.2 ml collagen, or at 24 h
with 0.1 or 0.2 ml collagen. The influence of media type and additives was examined in hepatocytes overlaid at 0 h with 0.2
ml collagen and incubated in DMEM+0.1μM DEX, DMEM+0.1μM DEX+5% FBS, Williams' medium E+0.1μM DEX+1% ITSΘ+, DMEM +1.0μM DEX, DMEM+1.0 μM DEX+5% FBS, or modified Chee's medium (MCM)+0.1 μM DEX+1% ITSГ+. [3H] TC accumulation by hepatocytes in Hank's balanced salt solution (HBSS) and Ca2+-free HBSS was measured, and the biliary-excretion index (BEI: percentage of accumulated TC localized in the canalicular compartment)
was calculated. Light microscopy and carboxydichlorofluorescein fluorescence were employed to examine the cellular and canalicular
morphologies. The volume of collagen used for both the substratum and the overlay did not affect TC accumulation or biliary
excretion. The BEI tended to be higher in cells overlaid at 24 h (BEI=0.649 [0.1 ml collagen]; BEI=0.659 [0.2 ml collagen])
compared with those overlaid at 0 h after seeding (BEI=0.538 [0.1 ml collagen]; BEI=0.517 [0.2 ml collagen]), although the
differences were not statistically significant. Hepatocytes cultured in MCM produced consistently the lowest BEI of TC (BEI=0.396).
Differing DEX concentration (0.1 μM versus 1.0 μM) with or without 5% FBS did not appear to have a significant effect on the BEI of TC. 相似文献
18.
We describe a protocol for somatic embryogenesis of Protea cynaroides, with potential for high frequency production of this important horticultural species. Somatic embryos formed directly on
both P. cynaroides mature zygotic embryos and excised cotyledons cultured on MS medium without growth regulators. The addition of growth regulators
such as naphthalene acetic acid (NAA) (5; 13 and 27 μM) and 2,4-dichlorophenoxyacetic acid (2,4-D) (5; 11 and 23 μM), in combination
with thidiazuron (TDZ) (1 μM), benzylaminopurine (BAP) (1 μM) or kinetin (1 μM) suppressed the formation of somatic embryos.
After eight weeks in culture, formation of somatic embryos was observed. Zygotic explants formed the most embryos when cultured
in a 12-h photoperiod in comparison to explants cultured in the dark. Up to 83% of these embryos germinated after transferal
to the germination medium containing 0.3 μM GA3. Significantly fewer embryos germinated in MS medium with no growth regulators, or supplemented with higher concentrations
of GA3, while low germination percentages were also observed in MS media containing casein hydrolysate and coconut water. The germination
of normal somatic embryos (two separate cotyledons and a single radicle) was observed only in media containing either no growth
regulators, 0.3 μM GA3 or 1 μM GA3. All embryos that germinated in high concentrations of GA3 were malformed. 相似文献
19.
Koichi Hirata Tadashi Oku Aaron E. Freeman 《In vitro cellular & developmental biology. Plant》1982,18(9):789-799
Summary Twenty to twenty-two days postcoitum mouse fetal pancreas organ bits were cultured on the dermal surface of irradiated pigskin
as a substrate. The medium used for long term culture consisted of Eagle’s Minimum Essential Medium with the addition of 10%
bovine serum, 0.02 U/ml insulin, 0.025 μg/ml glucagon, 3.63 μg/ml hydrocortisone, 100 μg/ml soybean trypsin inhibitor or 10−8
M atropine. When the medium lacked trypsin inhibitor or atropine but contained the three hormones, the pigskin support began
to be destroyed after 2 to 4 wk in culture. Thereafter, the cultured cells could not grow and survive on the digested pigskin.
When 10−6
M atropine was added to the medium, amylase secretion from cultured cells and destruction of pigskin were inhibited completely
but pancreas cells could not grow or survive. In contrast, 100 μg/ml soybean trypsin inhibitor or 10−8
M atropine permitted cell growth, permitted amylase secretion from the cultured acinar cells, and prevented the destruction
of pigskin. Under these conditions pancreas cells migrated or grew or both from the organ bits onto the surface of the pigskin
dermis and organoid aggregations formed. Hydrocortisone was needed to permit growth for more than 2 wk. Glucagon and insulin
had additive effects. Light and electron microscopic observations indicated the culture of at least five kinds of cells, i.e.,
duct, acinar, centroacinar, endocrine, and mesenchymal. The majority of cultured cells were duct cells and acinar cells. There
were few mesenchymal cells. Mouse pancreas cells were cultured for at least 12 wk by this method.
This investigation was supported by PHS Grant CA 30220 awarded by the National Cancer Institute, DHHS, Grant 1203M awarded
by the Council for Tobacco Research, Inc., and Grant RD-65 (for equipment) awarded by the American Cancer Society. Nude mice
were provided by Dr. Wendall M. Farrow of Life Sciences, Inc., Resource Laboratory N01, CP6-1005 of the National Cancer Institute. 相似文献
20.
A novel protocol for indirect shoot organogenesis of Dieffenbachia cv. Camouflage was established using leaf explants excised from in vitro shoot cultures. The frequency of callus formation
reached 96% for explants cultured on Murashige and Skoog (1962) basal medium supplemented with 5 μM thidiazuron and 1 μM 2,4-dichlorophenozyacetic acid. The number of shoots regenerated
was high, with up to 7.9 shoots produced per callus cultured on basal medium supplemented with 40 μM N
6-(Δ2-isopentenyl)adenine and 2 μM indole-3-acetic acid. Regenerated shoots rooted well in a soilless substrate, acclimatized ex
vitro at 100%, and grew vigorously under shaded greenhouse conditions. Somaclonal variations in leaf variegation, color, and
morphology have been observed in regenerated plants. 相似文献