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1.
We compared the effects of four quaternary benzo[c]phenanthridine alkaloids – chelerythrine, chelilutine, sanguinarine, and sanguilutine – and two quaternary protoberberine
alkaloids – berberine and coptisine – on the human cell line HeLa (cervix carcinoma cells) and the yeastsSaccharomyces cerevisiae andSchizosaccharomyces japonicus var. versatilis. The ability of alkaloids to display primary fluorescence, allowed us to record their dynamics and localization in cells.
Cytotoxic, anti-microtubular, and anti-actin effects in living cells were studied. In the yeasts, neither microtubules nor
cell growth was seriously affected even at the alkaloid concentration of 100 μg/ml. The HeLa cells, however, responded to
the toxic effect of alkaloids at concentrations ranging from 1 to 50 μg/ml. IC50 values for individual alkaloids were: sanguinarine IC50 = 0.8 μg/ml, sanguilutine IC50 = 8.3 μg/ml, chelerythrine IC50 = 6.2 μg/ml, chelilutine IC50 = 5.2 μg/ml, coptisine IC50 = 2.6 μg/ml and berberine IC50 >10.0 μg/ml. In living cells, sanguinarine produced a decrease in microtubule numbers, particularly at the cell periphery,
at a concentration of 0.1 μg/ml. The other alkaloids showed a similar effect but at higher concentrations (5–50 μg/ml). The
strongest effects of sanguinarine were explained as a consequence of its easy penetration through the cell membrane owing
to nonpolar pseudobase formation and to a high degree of molecular planarity.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
2.
Thomas S. Vates Garry L. Brake S. K. Majumdar Gail L. Ferguson 《In vitro cellular & developmental biology. Plant》1986,22(6):305-310
Summary Cis-diamminedichloroplatinum II (cis-DDP), an antitumor drug and the inactive trans-isomer were studied to evaluate their
effects on cell multiplication, DNA synthesis, and surface morphology of the murine erythroleukemia cells (clone 6A11A). Short-term
treatment of cells (1h) with 5 and 10μg/ml of cis-DDP resulted in a significant inhibition of cell multiplication. Continuous
treatment with cis-DDP (up to 144 h) significantly arrested cell growth at 1,5, and 10μg/ml. The cells exposed to 10 μg/ml
trans-DDP exhibited a slight decrease in cell multiplication; however, the 25-μg/ml treatments showed a modest inhibition
of cell growth. Continuous treatment with cis-DDP resulted in a concentrationdependent decrease in DNA synthesis, although
low-dose treatment (0.05 and 0.1 μg/ml), with a few exceptions, had no relative inhibitory effect. Likewise, trans-DDP treatments
decreased tritiated thymidine incorporation; however, this inhibitory effect was not as drastic as with corresponding concentrations
of cis-DDP. Scanning electron microscope studies revealed the formation of many giant cells and blebs at all short-term treatment
concentrations of cis-DDP past the 48 h interval. Continuous treatment of cis-DDP at 1 μg/ml concentration produced giant
cells with minute holes, whereas the 5 and 10 μg/ml exposure resulted in the formation of blebs and large holes and reduction
of microvilli past the 48-h treatment period. At higher concentrations the continuous treatment of cis-DDP completely destroyed
the cells. The surface morphology of trans-isomer treated cells, in most instances, resembled the corresponding untreated
control cells. 相似文献
3.
An electrophysiological freeze fracture assessment of cadmium nephrotoxicity in vitro 总被引:1,自引:0,他引:1
Debra J. Hazen-Martin Donald A. Sens John G. Blackburn Mary C. Flath Mary Ann Sens 《In vitro cellular & developmental biology. Plant》1989,25(9):791-799
Summary Human proximal tubule cell cultures exposed to doses of cadmium chloride (CdCl2) between 0.05 μg/ml and 0.5 μg/ml exhibited alterations in cell membrane structure and transport function. At these Cd concentrations,
cell numbers were not significantly altered from control values in either nonreplicating confluent, or actively replicating
subconfluent cultures. Transmission electron microscopy revealed few alterations in cultures treated with 0.05 μg/ml Cd. Tight
junctions were intact; organelles and myeloid body formation appeared normal. Freeze fracture analysis confirmed the integrity
of the tight junctions as well as increased numbers of vesicles or pits along the lateral cell membrane, indicating increased
endocytotic activity. Cells exposed to 0.1 μg/ml Cd were characterized by decreased numbers of microvilli and inhibited myeloid
body formation. Cd doses of 0.5 μg/ml elicited nuclear chromatin condensation, fragmented sealing strands in 5 to 10% of the
tight junction profiles, sparse microvilli, and inhibited myeloid body formation. Electrophysiologic assessments of transport
function by Ussing chamber analysis revealed decreases in transepithelial potentials for all three concentrations, with significant
differences at Cd concentrations of 0.5 to 0.1 μg/ml. Cells treated with 0.5 μg/ml Cd also exhibited slight decreases in electrical
resistance, consistent with the minimal fragmentation of sealing strands observed in freeze fracture replicas. Resistance
in cultures treated with 0.1 or 0.05 μg/ml Cd remained within control values and indicated that drops in potential difference
and short circuit current in these cells reflected true alterations in ion transport.
This paper was presented at a Symposium on the Physiology and Toxicology of the Kidney In Vitro co-sponsored by The Society
of Toxicology (SOT) and the Tissue Culture Association field at the 27th annual meeting of the SOT in Dallas, Texas in 1988.
This work was supported by the Johns Hopkins Center for Alternatives to Animal Testing. The Balzers Freeze Fracture Unit utilized
in these studies was provided by equipment grant S10 RR02329 from the National Institutes of Health, Bethesda, MD. 相似文献
4.
The effect of noncytotoxic doses of argemone oil (AO) and butter yellow (BY), the common adulterants in edible oil, on free
radical generation and signaling pathway for cell proliferation in primary cells of gall bladder (GB) was undertaken. AO and
BY showed no cytotoxicity at 0.1 μl/ml and 0.1 μg/ml concentration, respectively. AO caused significant increase in ROS after
30 min and RNS after 24 h in GB cells while no change was observed following BY treatment. Enhanced level of COX-2 was observed
following AO (0.1 μl/ml) and BY (0.1 μg/ml) treatment to cells for 24 h. AO treatment caused phosphorylation of ErbB2, AKT,
ERK, and JNK along with increased thymidine uptake indicating cell proliferation ability in GB cells. BY treatment also showed
significant expression of these proteins with the exception of phosphorylated JNK. These results suggest that AO and BY have
cell proliferative potential in GB cells following up-regulation of COX-2 and ErbB2; however, their downstream signaling molecules
and free radical generation have differential response, indicating that the mechanism of proliferation is different for both
compounds and may have relevance in gall bladder cancer. 相似文献
5.
Summary A simple technique which results in good quality early mitotic stages of amniotic fluid (AF) cells is presented. Two days after trypsinization AF cell cultures are incubated for 4 h in culture medium containing 20 U/ml liquemin. During the last hour 5 g/ml ethidium bromide (EB) is added and 15 min before harvest 0.04 g/ml colcemid is applied as usual. G-banded and Q-banded chromosomes corresponding to at least 550–850 bands per haploid genome can be obtained in sufficient numbers. 相似文献
6.
Ursula J. Behrens Fiorenzo Paronetto 《In vitro cellular & developmental biology. Plant》1984,20(5):391-395
Summary In our laboratory, airborne yeast contaminants of cell cultures have consistently been of the genusCandida (speciesCandida parapsilosis), which are difficult to control with fungicidal agents. To salvage cell lines that show the presence of this fungus, two
effective methods may be employed. In early stages of infection, the addition of activated mouse peritoneal macrophages (5×105 cells/ml) to the culture medium containing 5 μg Fungizone/ml eliminates all spores by phagocytosis. More heavily contaminated
cultures can be depleted of fungi by density centrifugation on a layer of 38% Percoll. Remaining single spores, often not
detectable by light microscopy, can be removed by the addition of macrophages (2×105/ml) and Fungizone (5 μg/ml) to the culture medium. Contaminated monolayer cells can be freed of blastospores by several washes
with balanced salt solution and subsequent culturing for 4 d in medium containing 10 μg Fungizone/ml without any toxic effects
to the cells. These procedures can rescue valuable cell lines and hybridomas that would otherwise be lost.
This work was supported by Veterans Administration Research Funds. 相似文献
7.
Thymineless death (TLD) was studied inLactobacillus acidophilus R-26. Thymine synthesis was inhibited with 5-fluorouracil (FU) or deoxyadenylate (dAMP) or by the absence of folic acid.
In the case of FU, the maximum rate of dying was obtained at concentrations exceeding 0.1 μg/ml. This concentration did not
affect the growth of the bacteria in the presence of thymine (4 μg/ml) and uracil (10 μg/ml). At higher FU concentrations
up to 10 μg/ml, the course of TLD was unaltered, but the growth of bacteria in complete medium was slower. In the case of
dAMP, the same course of TLD was obtained at a concentration of 150 μg/ml. If 1,500 μg dAMP/ml was used, the pre-death lag
phase was shortened the rate of dying being unaltered. These concentrations of dAMP retarded the growth of bacteria even in
a complete medium. If the thymine synthesis was prevented by the absence of folic acid the rate of dying was much lower than
that caused by the presence of FU or dAMP. This was true even if the aminopterin was added. The authors conclude that the
folic acid starvation did not inhibit completely the synthesis of thymine. 相似文献
8.
A method for isolating large numbers of viable disaggregated cells from various human tissues for cell culture establishment 总被引:4,自引:0,他引:4
Ruth E. Gibson-D'ambrosio Mervyn Samuel Steven M. D'Ambrosio 《In vitro cellular & developmental biology. Plant》1986,22(9):529-534
Summary A method is described for the isolation of large numbers of viable disaggregated cells from human tissues. This method combined
the mechanical action of a Stomacher Model 80 Lab Blender, 0.1 mg/ml trypsin or 0.5 mg/ml collagenase, and 0.1 mM [ethylene bis(oxyethylenenitrolo)]-tetraacetic acid (EGTA). Tissue (0.2 to 1.0 g) obtained from human fetal intestine, kidney,
liver, lung, and skin were separately minced into approximately 1-mm3 pieces. The pieces were placed in a sterile bag containing 60 ml of calcium- magnesium-free phosphate buffered saline, the
appropriate enzyme (0.1 mg/ml trypsin or 0.5 mg/ml collagenase) plus 0.1 mM EGTA, and 0.1% methylcellulose. The bag was then placed into the blender and mixed at a low speed for 3 to 20 min at room
temperature. After a single cell suspension was observed by phase contrast microscopy, 10 ml of bovine calf serum was added
to the cell suspension to inactivate the proteolytic enzymes. At this time 130 ml of cold Hanks' balanced salts solution containing
5% bovine calf serum was added and the entire cell suspension passed through a tissue sieve (100 mesh, 140 μm) and the cells
collected by centrifugation. These cells were then resuspended into the appropriate culture medium. In comparison to other
methods for establishment of cell cultures from human tissues, the method described requires shorter incubation times with
relatively low concentrations of proteolytic enzymes, and yields two- to three-fold greater number of cells per tissue with
86 to 93% viability. Also, depending on the cell type, 50 to 75% of the isolated cells attached to the culture vessel within
24 h. Variation of the time and concentration of digestive enzymes can be used to select different cell types for culture.
This work was supported by research grants from the National Institute of Environmental Health Sciences, Bethesda, MD (ES3101)
and the United States Environmental Protection Agency, Washington, D. C. (R810146). 相似文献
9.
Hematopoietic progenitor colony assays were used to establish the effects of the vinca alkaloid vinorelbine (VRB) on murine
bone marrow. The in vitro growth of colony-forming units–granulocyte/macrophage (CFU-GM), burst forming units–erythroid (BFU-E) and colony-forming
units–mix (CFU-mix) was dose-dependently inhibited by VRB. The highest dose assayed (0.02 μg/ml) suppressed all of the different
progenitor cells by 100%. A comparison of the dose–response curves showed that CFU-GM, BFU-E, and CFU-mix exhibited similar
patterns of sensitivity to the cytotoxic action of VRB. Long-term bone marrow cultures have provided a valuable in vitro model for studying the role of the microenvironment of bone marrow. Cellularity of stromal layers was reduced with increasing
doses of VRB. The appearence of these layers was altered minimally with the lowest dose used; a gradual loss of cellularity
was seen in cultures exposed to 0.05 and 0.075 μg/ml; and a marked loss at the dose of 0.1 μg/ml. Our results show that VRB
has an important effect on hematopoietic progenitors at the highest dose tested, while the stromal cells were not affected
at a similar dose (0.025 μg/ml), suggesting that the stroma is more resistant to this drug.
This revised version was published online in July 2006 with corrections to the Cover Date. 相似文献
10.
Natural killer (NK) cells are non-T, non-B cell lymphocytes that lyse a variety of tumor and virus-infected cells. In this
study, we demonstrated that phytohemagglutinin (PHA) rendered resistant autologous T cells extremely sensitive to natural-killer(NK)-cell-mediated
lysis. The sensitization was very rapid and concentration-dependent (0.01–1 μg/ml); 62% and 95% of autologous T cells were
lysed by interleukin-2-activated NK cells 5 min and 18 h respectively after treatment with PHA (1 μg/ml). The maximal decrease
in the level of MHC class I molecules observed on T cells was 22%. Induction of susceptibility to NK-mediated lysis was correlated
with the expression of activation markers on T cells treated for relatively long intervals (more than 18 h) with high concentrations
of PHA (more than 0.1 μg/ml). Sensitization of T cells required RNA and protein synthesis, although DNA synthesis was not
essential. We propose that this unique system is suitable for studying the mechanisms involved in recognition and killing
of target cells by NK cells.
Received: 11 February 1999 / Accepted: 28 July 1999 相似文献
11.
Yasuhiro Tomooka Stephen E. Harris John A. McLachlan 《In vitro cellular & developmental biology. Plant》1985,21(4):237-244
Summary Epithelial cells from mouse seminal vesicles were enzymatically dissociated enriched by gradient centrifugation, and maintained
in collagen gel cultures with defined (serum-free) media. The epithelial origin of the cells was determined morhologically,
immunocytochemically, and biochemically. Cells formed three-dimensional colonies with a lumen in collagen gels. Cell number
was increased eight-fold within a 8 to 12-d culture period in a medium supplemented with epidermal growth factor (EGF) (10
ng/ml), insulin (10 μg/ml), transferrin (10 μg/ml), cholera toxin (10 ng/ml), and hydrocortisone (0.1 μg/ml). The cells required
eGF and insulin; the growth-promoting effects of these two peptide hormones were optimized by transferrin, cholera toxin,
and hydrocortisone. Fetal bovine serum did not support growth; rather, it suppressed the stimulated growth observed in serum-free
media. A time-course study revealed that a lag period preceded rapi growth. The collagen gel, serum-free culture provides
a powerful tool to study the effects of hormones on proliferation and differentiation of androgen sensitive cells. 相似文献
12.
F. L. Vaughan L. L. Kass J. A. Uzman 《In vitro cellular & developmental biology. Plant》1981,17(11):941-946
Summary A procedure for the preparation and cultivation of rat epidermal basal cells from full thickness skin resulted in greater
than 99% viability and 90% plating efficiency. However, attempts to subculture monolayers of these epithelial cells grown
in medium with serum as the only supplement were totally unsuccessful. When hydrocortisone and insulin were added to the medium,
subcultivation of primary growth was obtained. It was demonstrated that hydrocortisone at concentrations as low as 0.1 μg/ml
was necessary for at least the initial attachment of the cells to the substrate—an essential step in subcultivation. Increasing
concentrations of insulin (0.1 to 50 μg/ml) caused the rate of proliferation and the cell density to increase, but insulin
alone did not support subcultivation.
This work was supported by Grants 1-R01-CA-19988 and 5-R01-AM-15206 from the National Institutes of Health, U.S. Public Health
Service. 相似文献
13.
CSD mRNA expression in rat testis and the effect of taurine on testosterone secretion 总被引:1,自引:0,他引:1
Jiancheng Yang Gaofeng Wu Ying Feng Changmian Sun Shumei Lin Jianmin Hu 《Amino acids》2010,39(1):155-160
In the present study, the cysteine sulfinate decarboxylase (CSD) mRNA expression was detected in rat testis by RT-PCR. The
results showed that CSD mRNA was expressed in rat testis, and the putative encoded-amino acid sequence was exactly the same
as that in rat liver which was already known. At the same time, the effects of taurine on testosterone secretion were investigated
both in vivo and in vitro. In vivo, taurine were administered to male rats by tap water. The results showed that taurine obviously
stimulated the secretion of FSH, LH and testosterone in serum, but showed no significant effect on the secretion of estradiol.
Taurine administered in water could significantly increase the concentration of taurine in the blood and testis of rats. In
vitro, cultured Leydig cells were treated with taurine independently or incubated with human chorionic gonadotropin (HCG)
and progesterone. The results showed that taurine had biphasic effects on basal testosterone secretion in cultured Leydig
cells. Low concentrations of taurine (0.1–100 μg/ml) could stimulate testosterone secretion, whereas high concentration of
taurine (400 μg/ml) could inhibit testosterone secretion. Testosterone secretion stimulated by HCG was significantly increased
by 10 and 100 μg/ml of taurine administration, and obviously decreased by treating with 400 μg/ml of taurine. Testosterone
secretion induced by progesterone was significantly stimulated by treating with 1.0 and 10 μg/ml of taurine, however, it was
significantly inhibited when treated with 400 μg/ml of taurine. Meanwhile, the effect of silencing CSD mRNA by siRNA on testosterone
secretion was analyzed. The results showed that testosterone secretion was obviously decreased after the inhibition of CSD
mRNA expression in cultured Leydig cells. These results indicated that taurine can be synthesized in rat testis by CSD pathway,
and it plays important roles in testosterone secretion both in vivo and in vitro which need to be further investigated. 相似文献
14.
Don J. Brenner Hervé Bercovier Jan Ursing Jean Michel Alonso Arnold G. Steigerwalt G. Richard Fanning Geraldine P. Carter H. H. Mollaret 《Current microbiology》1980,3(4):207-212
The drugs griseofulvin (10 μg/ml), nalidixic acid (0.05 μg/ml), quinine dihydrochloride (50 μg/ml), quinine ethylcarbonate
(50 μg/ml), quinine urea hydrochloride (50 μg/ml), quinine lactate (50 μg/ml), and pamaquine (50 μg/ml) were chosen for laboratory
studies. The minimal inhibitory concentration of the drug was used for determining the range of drug concentration needed
to produce “mutational synergism” with ultraviolet radiation. Forward mutation from streptomycin sensitivity to resistance
was used as a marker for mutagenicity. No stimulatory or inhibitory effects were noted on viable counts and mutation frequency,
when the drugs were added (20–60 μg/ml) to the growth medium of unirradiatedEscherichia coli HCR+, HCR−, and irradiated HCR− strains. These drugs increased mutation frequency and lethality of irradiated HCR+ bacteria. Incorporation of adenine (6 μm) into the minimal expression medium reverses the mutagenic effect of chloroquine.
Chloroquine (50 μg/ml) did not interfere with the photoactivation of irradiated HCR+ cells. Our findings suggest that these chemicals selectively interfere with excision-repair. 相似文献
15.
Masahiro Matsuda Shiro Shigeta Koichi Okutani 《Marine biotechnology (New York, N.Y.)》1999,1(1):68-73
A marine Pseudomonas species WAK-1 strain simultaneously produces extracellular glycosaminoglycan and sulfated polysaccharide. Among the antiviral
activities tested for these polysaccharides, the latter showed anti-HSV-1 activity in RPMI 8226 cells (50% effective concentration
is 1.4 μg/ml). Oversulfated derivatives of these polysaccharides prepared by dicyclohexylcarbodiimide-mediated reaction for
both polysaccharides showed antiviral activities against influenza virus type A (for glycosaminoglycan, 50% effective concentration
is 11.0 μg/ml; for another, 2.9 μg/ml). Glycosaminoglycan, sulfated polysaccharide, and their chemically synthesized oversulfated
derivatives did not show antiviral activities against influenza virus type B and human immunodeficiency virus type 1. No cytotoxicity
of these products was noted against host cells at the 50% cytotoxic concentration of 100 μg/ml, except that naturally occurring
sulfated polysaccharide had 50% cytotoxicity against MT-4 cells at 8–21 μg/ml.
Received May 1, 1998; accepted July 24, 1998. 相似文献
16.
Ming-Sound Tsao Judith D. Smith Joe W. Grisham 《In vitro cellular & developmental biology. Plant》1985,21(5):249-253
Summary The ability of a normal rat liver epithelial cell line with phenotypic characteristics of “oval” cells to grow in calcium-poor
medium has been investigated. The growth of these cells could be arrested in medium containing 0.03 mM Ca2+, a concentration below which cell necrosis began to occur 24 h postexposure. With increasing calcium concentration, progressive
cell proliferation was observed. Epithelial growth factor (EGF) (10 ng/ml) increased the survival and proliferation of cells
in calcium-poor medium and the response was inversely correlated with the extracellular calcium concentration. In contrast,
phenobarbital (0.2 to 2 mM), 12-0-tetradecanoylphorbol-13-acetate (0.01 to 1 μg/ml), or retinoic acid (0.001 to 0.1 μg/ml) depressed growth of cells
in calcium-poor medium. The results confirm the ability of EGF to lower the calcium requirement for proliferation of normal
cells, but such an effect does not seem to be a universal property of tumor promoters.
This research was supported by National Institutes of Health Grant CA 29323. 相似文献
17.
Growth of MTW9/PL2 estrogen-responsive rat mammary tumor cells in hormonally defined serum-free media 总被引:3,自引:0,他引:3
David Danielpour Terry L. Riss Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(1):42-52
Summary Growth of the estrogen-responsive MTW9/PL2 rat mammary tumor cells was demonstrated in serum-free defined medium (designated
DDM-1) formulated with F12-DME (1:1 vol/vol) supplemented with 15 mM HEPES pH 7.4 insulin 10 μg/ml, transferrin 10 μg/ml, sodium selenite 10 ng/ml, triiodo-l-thyronine 0.3 nM, phosphoethanolamine 5 μM, epidermal growth factor (20 ng/ml), 17 β-estradiol 2 nM, and bovine serum albumin 20 μg/ml. In DDM-1, the growth rate was about one-half that seen in serum-containing medium. When
ethanolamine (50 μM), glutathione (20 μg/ml), and linoleic acid/bovine serum albumin (150 μg/ml) were added (formulation DDM-2), the growth rate
was 80% of serum-containing medium and not seed-density dependent. Deletion of estradiol from DDM-1 or DDM-2 had no effect
on growth rate. Also, cells grown in steroid hormone deficient medium for 4 mo. continued to form estrogen-responsive tumors
in rats as did cells cultured for the same period in 2 nM estradiol. To investigate autocrine growth factor secretion, a third medium (DDM-3) was prepared by deleting insulin, epidermal
growth factor, phosphoethanolamine, estradiol, and both forms of bovine serum albumin from DDM-2. Growth in mitogen-free medium
equaled 86% of the serum-stimulated rate and was seed-density dependent; phenol red deletion from DDM-3 had no effect on growth
rate. Evidence presented suggests that autocrine factors stimulate growth of the MTW9/PL2 cells in DDM-3, and that this secretion
may support the growth of estrogen-responsive cells in culture in the absence of steroid hormone.
This work was supported by National Cancer Institute grants CA-38024 and CA-26617 and American Cancer Society grant BC255. 相似文献
18.
The extracellular metalloprotease (SMP 6.1) produced by a soil isolate of Serratia marcescens NRRL B-23112 was purified and characterized. SMP 6.1 was purified from the culture supernatant by ammonium sulfate precipitation,
acetone fractional precipitation, and preparative isoelectric focusing. SMP 6.1 has a molecular mass of approximately 50 900 Da
by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). The following substrates were hydrolyzed: casein,
bovine serum albumin, and hide powder. SMP 6.1 has the characteristics of a metalloprotease, a pH optimum of 10.0, and a temperature
optimum of 42° C. The isoelectric point of the protease is 6.1. Restoration of proteolytic activity by in-gel renaturation
after SDS-PAGE indicates a single polypeptide chain. SMP 6.1 is inhibited by EDTA (9 μg/ml) and not inhibited by antipain
dihydrochloride (120 μg/ml), aprotinin (4 μg/ml), bestatin (80 μg/ml), chymostatin (50 μg/ml), E-64 (20 μg/ml), leupeptin
(4 μg/ml), Pefabloc SC (2000 μg/ml), pepstatin (4 μg/ml), phosphoramidon (660 μg/ml), or phenylmethylsulfonyl fluoride (400
μg/ml). SMP 6.1 retains full activity in the presence of SDS (1% w/v), Tween-20 (1% w/v), Triton X-100 (1% w/v), ethanol (5%
v/v), and 2-mercaptoethanol (0.5% v/v). The extracellular metalloprotease SMP 6.1 differs from the serratiopeptidase (Sigma)
produced by S. marcescens ATCC 27117 in the following characteristics: isoelectric point, peptide mapping and nematolytic properties.
Received: 22 November 1996 / Received revision: 27 February 1997 / Accepted: 7 March 1997 相似文献
19.
Karolina Čepurnienė Paulius Ruzgys Rimantas Treinys Ingrida Šatkauskienė Saulius Šatkauskas 《The Journal of membrane biology》2010,236(1):81-85
DNA electrotransfer in vivo for gene therapy is a promising method. For further clinical developments, the efficiency of the
method should be increased. It has been shown previously that high efficiency of gene electrotransfer in vivo can be achieved
using high-voltage (HV) and low-voltage (LV) pulses. In this study we evaluated whether HV and LV pulses could be optimized
in vitro for efficient DNA electrotransfer. Experiments were performed using Chinese hamster ovary (CHO) cells. To evaluate
the efficiency of DNA electrotransfer, two different plasmids coding for GFP and luciferase were used. For DNA electrotransfer
experiments 50 μl of CHO cell suspension containing 100, 10 or 1 μg/ml of the plasmid were placed between plate electrodes
and subjected to various combinations of HV and LV pulses. The results showed that at 100 μg/ml plasmid concentration LV pulse
delivered after HV pulse increased neither the percentage of transfected cells nor the total transfection efficiency (luciferase
activity). The contribution of the LV pulse was evident only at reduced concentration (10 and 1 μg/ml) of the plasmid. In
comparison to HV (1,200 V/cm, 100 μs) pulse, addition of LV (100 V/cm, 100 ms) pulse increased transfection efficiency severalfold
at 10 μg/ml and fivefold at 1 μg/ml. At 10 μg/ml concentration of plasmid, application of four LV pulses after HV pulse increased
transfection efficiency by almost 10-fold. Thus, these results show that contribution of electrophoretic forces to DNA electrotransfer
can be investigated in vitro using HV and LV pulses. 相似文献
20.
Masami Ogasawara David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1988,24(9):911-920
Summary Growth of the MCF-7, T47D, and ZR-75-1 human breast cancer cells was established in a serum-free defined medium (MOM-1) composed
of a 1∶1 (vol/vol) mixture of Ham's F12 medium and Dulbecco's modified Eagle's medium containing 15 mM HEPES (pH 7.2), 2 mM 1-glutamine, 20 μg/ml glutathione, 10 μg/ml insulin, 10 μg/ml transferrin (Tf), 10 ng/ml selenous acid, 0.3 nM triiodothyronine, 50 μg/ml ethanolamine, 20 ng/ml epidermal, growth factor, 2.0 nM 17β-estradiol, and 1.0 mg/ml bovine serum albumin (BSA). Proliferation in MOM-1 was 50 to 70% of the serum stimulated rate.
Deletion of components from MOM-1 gave a medium (Tf-BSA) containing only HEPES, 10 μg/ml Tf, and 200 μg/ml BSA, which sustained
MCF-7 and T47D cells in a slowly dividing and mitogen responsive state; ZR-75-1 cells required Tf plus 1.0 mg/ml BSA. In Tf-BSA,
insulin and insulin-like growth factor I(IGF-I) were mitogenic with ED50 values of 2 to 3 ng/ml and 30 to 150 pg/ml, respectively, with MCF-7 cells. The T47D cells were responsive to these factors
in Tf-BSA but required 10-fold higher concentrations for ED50. At saturating concentrations, insulin and IGF-I promoted 1.5 to 3.5 cell population doublings over controls in 8 d. At≤ng/ml
concentrations, epidermal growth factor, insulin-like growth factor II, and basic fibroblast growth factor were mitogenic
for human breast cancer cells in Tf-BSA. Mitogen activities in uterus and pituitary extracts were assayed readily in Tf-BSA.
This new method offers a convenient means of comparing the potencies of growth-promoting factors on human breast cancer cells
without interfering activities known to be present in serum.
This work was supported by grants CA-38024 and CA-26617, from the National Cancer Institute, Bethesda, MD, and by American
Cancer Society grant BC-255 and grant 2225 from the Council for Tobacco Research, USA, Inc. 相似文献