共查询到20条相似文献,搜索用时 31 毫秒
1.
Thomas P. Dousa 《Cell biochemistry and biophysics》1998,29(1-2):19-34
Mesangial cells (MC) of renal glomeruli respond to immune-inflammatory injury by accelerated proliferation and generation
of reactive oxygen metabolites (ROM). We studied in vivo and in vitro roles of cAMP-protein kinase A (PKA) signaling in modulation
of these pathobiologic processes with focus on PDE isozymes. Mitogenic synthesis of DNA in mesangial cells grown in primary
culture was blocked by forskolin and dibutyryl cyAMP. Incubation of MC with PDE-3 inhibitors, cilostamide and lixazione, inhibited
(>50%) mitogenesis, whereas inhibitors of PDE-4, rolipram and denbufylline, caused little or no inhibition. Conversely, inhibitors
of PDE-4 suppressed generation of ROM in MC, whereas inhibitors of PDE-3 had no effect. Incubation of mesangial cells with
cilostamide or with rolipram increasedin situ activity of PKA, and effects of the two inhibitors were additive. PDE inhibitors also decreased activity of mitogen-activated
protein kinase. The efficacy of PDE isozyme inhibitors (IC50) to suppress mitogenesis or ROM generation paralleled IC50 for inhibition of cAMP hydrolysis by extracts from mesangial cells. Administration of lixazinone or lixazione in combination
with rolipram to rats with mesangial proliferative glomerulonephritis induced by antithymic serum suppressed proliferation
of mesangial cells and also reduced other histopathologic manifestations of the disease. Based on these observations, we propose
that in MC, a cAMP pool that is hydrolyzed by PDE-3 inhibits by negative crosstalk via activation of PKA, mitogen-activated
protein kinase (MAPK) pathway, and mitogenesis; whereas cAMP pool linked to PDE-4 inhibits, also via activation of PKA, ROM
generation in mesangial cells. Results also suggest that PDE isozyme inhibitors, in particular inhibitors of PDE-3, should
be investigated for potential use for “signal transduction pharmacotherapy” of glomerulonephritis. 相似文献
2.
M. J. Perry J. O’Connell C. Walker T. Crabbe D. Baldock A. Russell S. Lumb Z. Huang D. Howat R. Allen M. Merriman J. Walls T. Daniel B. Hughes F. Laliberte G. A. Higgs R. J. Owens 《Cell biochemistry and biophysics》1998,29(1-2):113-132
We present the in vitro characterization of a novel phosphodiesterase type 4 inhibitor, CDP840 (R-[+]-4-[2-{3-cyclopentyloxy-4-methoxyphenyl}-2-phenylethyl]pyridine), which has shown efficacy in a phase II allergen challenge
study in asthmatics without adverse effects. CDP840 potently inhibits PDE-4 isoenzymes (IC50 2–30 nM) without any effect on PDE-1, 2, 3, 5, and 7 (IC50>100 μM). It exhibited no significant selectivity in inhibiting human recombinant isoenzymes PDE-4A, B, C or D and was equally active
against the isoenzymes lacking UCR1 (PDE-4B2 and PDE-4D2). In contrast to rolipram, CDP840 acted as a simple competitive inhibitor
of all PDE-4 isoenzymes. Studies with rolipram indicated a heterogeneity within all the preparations of PDE-4 isoenzymes,
indicative of rolipram inhibiting the catalytic activity of PDE-4 with both a low or high affinity. These observations were
confirmed by the use of a PDE-4A variant, PDE-4A330–886, which rolipram inhibited with low affinity (IC50=1022 nM). CDP840 in contrast inhibited this PDE-4A variant with similar potency (IC50=3.9 nM), which was in good agreement with theK
d of 4.8 nM obtained from [3H]-CDP840 binding studies. Both CDP840 and rolipram inhibited the high-affinity binding of [3H]-rolipram binding to PDE-4A, B, C and D with similarK
d app (7–19 nM and 3–5 nM, respectively). Thus, the activity of CDP840 at the [3H]-rolipram binding site was in agreement with the inhibitor’s activity at the catalytic site. However, rolipram was ∼100-fold
more potent than CDP840 at inhibiting the binding of [3H]-rolipram to mouse brain in vivo. These data clearly demonstrate that CDP840 is a potent selective inhibitor of all PDE-4
isoenzymes. In contrast to rolipram, CDP840 was well-tolerated in humans. This difference, however, cannot at present be attributed
to either isoenzyme selectivity or lack of activity in vitro at the high-affinity rolipram binding site (Sr). 相似文献
3.
Jackie D. Corbin Alfreda Beasley Illarion V. Turko Tamara L. Haik Kimberly A. Mangum Jack N. Wells Sharron H. Francis Konjeti R. Sekhar 《Cell biochemistry and biophysics》1998,29(1-2):145-157
The cGMP-binding cGMP-specific phosphodiesterase (PDE-5) contains distinct catalytic and allosteric binding sites, and each
is cGMP-specific. Cyclic nucleotide phosphodiesterase inhibitors, such as 3-isobutyl-1-methylxanthine (IBMX), are believed
to compete with cyclic nucleotides at the catalytic sites of these enzymes, but the portion of PDE-5 that accounts for interaction
of either of these inhibitors or the substrates themselves with the catalytic domain of the enzymes has not been identified.
IBMX was derivatized to yield the photoaffinity probe 8([3-125I,-4-azido]-benzyl)-IBMX, which is referred to as 8(125IAB)-IBMX. This probe was incubated with partially purified recombinant bovine PDE-5. After UV irradiation and SDS-PAGE, a
single radiolabeled band that coincided with the position of PDE-5 was visualized on the gel, and the photoaffinity labeling
of PDE-5 was linear with increasing concentration of the 8(125IAB)-IBMX. Prominent Coomassie blue-stained bands other than PDE-5 were not labeled significantly. The photo-affinity labeling
was progressively blocked by cGMP at concentrations higher than 10 μM, whereas cAMP or 5′-GMP exhibited only weak inhibitory effects. Other compounds that are believed to interact with the PDE-5
catalytic site, including IBMX, clMP, and β-phenyl-1,N
2-etheno-cGMP (PET-cGMP), also inhibited the photoaffinity labeling in a concentration-dependent manner. The IC50 of PET-cGMP for inhibition of photoaffinity labeling was 10 μM, which compared favorably with an IC50 of 5 μM for inhibition of PDE-5 catalytic activity by this compound. It is concluded that the interaction of this photoaffinity probe
with PDE-5 is highly specific for the catalytic site over the allosteric binding sites of PDE-5 and could prove useful in
studies to map the catalytic site of PDE-5. 相似文献
4.
Rui He Narcisse Komas Dag Ekholm Taku Murata Masato Taira Steven Hockman Eva Degerman Vincent C. Manganiello 《Cell biochemistry and biophysics》1998,29(1-2):89-111
cDNAs encoding two PDE-3 or cyclic GMP-inhibited (cGI) cyclic nucleotide phosphodiesterase (PDE) isoforms, RPDE-3B (RcGIP1)
and HPDE-3A (HcGIP2), were cloned from rat (R) adipose tissue and human (H) heart cDNA libraries. Deletion and N- and C-terminal
truncation mutants were expressed inEscherichia coli in order to define their catalytic core. Active mutants of both RPDE-3B and HPDE-3A included the domain conserved among all
PDEs plus additional upstream and downstream sequences. An RPDE-3B mutant consisting of the conserved domain alone and one
from which the RPDE-3B 44-amino acid insertion was deleted exhibited little or no activity. All active recombinants exhibited
a high affinity (<1 μM) for cyclic AMP (cAMP) and cyclic GMP (cGMP), were inhibited by cAMP, cGMP, and cilostamide, but not by rolipram, and were
photolabeled with [32P]-cGMP. The IC50 values for cGMP inhibition of cAMP hydrolysis were lower for HPDE-3A than for RPDE-3B recombinants. The deduced amino acid
sequences of HPDE-3A and RPDE-3B catalytic domains are very similar except for the 44-amino acid insertion not found in other
PDEs. It is possible that this insertion may not only distinguish PDE-3 catalytic domains from other PDEs and identify catalytic
domains of PDE-3 subfamilies or conserved members of the PDE-3 gene family, but may also be involved in the regulation of
sensitivity of PDE-3s to cGMP.
These authors contributed equally to this work. 相似文献
5.
Alison M. Michie Graham Rena Margaret M. Harnett Miles D. Houslay 《Cell biochemistry and biophysics》1998,28(2-3):161-185
We have previously shown that the major cAMP phosphodiesterase (PDE) isoforms present in murine thymocytes are the cGMP-stimulated
PDE activity (PDE-2) and the cAMP-specific PDE activity (PDE-4), and that these isoforms are differentially regulated following
ligation of the TCR (Michie, A. M., Lobban, M. D., Mueller, T., Harnett, M. M., and Houslay, M. D. [1996]Cell. Signalling
8, 97–110). We show here that the anti-CD3-stimulated elevation in PDE-4 activity in murine thymocytes is dependent on protein
tyrosine kinase and protein kinase C (PKC)-mediated signals as the TCR-coupled increase in PDE-4 activity can be abrogated
by both the tyrosine kinase inhibitor, genistein, and the PKC selective inhibitors chelerythrine and staurosporine. Moreover,
the PKC-activating phorobol ester, phorbol-12-myristate, 13-acetate (PMA) caused an increase in PDE-4 activity, similar to
that observed in cells challenged with anti-CD3 monoclonal antibodies and which was not additive with cochallenge using anti-CD3
antibodies. Both the PMA-and the anti-CD3 antibody-mediated increases in PDE-4 activity were blocked by treatment with either
cycloheximide or actinomycin D. Despite the upregulation of PDE-4 activity consequent to TCR ligation, intracellular cAMP
levels increased on challenge of thymocytes with anti-CD3 antibody, indicating that adenylate cyclase activity was also increased
by TCR ligation. It is suggested that the anti-CD3-mediated increase in PDE-4 activity was owing to a rapid PKC-dependent
induction of PDE-4 activity following crosslinking of the TCR complex. This identifies “crosstalk” occurring between the PKA
and PKC signaling pathways initiated by ligation of the antigen receptor in murine thymocytes. That both adenylate cyclase
and PDE-4 activities were increased may indicate the presence of compartmentalized cAMP responses present in these cells. 相似文献
6.
《Cellular signalling》2002,14(3):277-284
PDE7A is a recently described 3′,5′-cyclic adenosine monophosphate (cAMP)-specific phosphodiesterase (PDE) whose expression has been detected in T-cells. As treatment with the methylxanthine theophylline, a nonspecific PDE inhibitor, induces apoptosis in leukemic cells from patients with the B-lineage malignancy chronic lymphocytic leukemia (CLL), we sought to determine if PDE7A was a target of theophylline therapy in such cells. Western analysis revealed expression of PDE7A in normal human splenic B-cells, primary CLL cells, and in a CLL-derived cell line (WSU-CLL). Among the six cAMP PDEs (PDE1B, PDE3B, PDE4A, PDE4B, PDE4D, and PDE7) examined in WSU-CLL, only PDE7A levels were augmented by treatment with methylxanthines. The activity of PDE7A isolated from the WSU-CLL cell line by immunoprecipitation was inhibited by theophylline and IBMX with IC50 values of 343.5 and 8.6 μM, respectively. WSU-CLL PDE7A was also up-regulated by a novel specific inhibitor (IC242), which inhibits PDE7A from WSU-CLL cells with an IC50 value of 0.84 μM. IC242-mediated up-regulation of PDE7A was blocked by the protein kinase A (PKA) inhibitor H-89. 相似文献
7.
Summary Methyl jasmonate (MeJA) interacted significantly with both indole-3-acetic acid (IAA) and 6-benzylaminopurine (BA) to influence
cell growth of cultured Onosma paniculatum cells. Cell growth decreased with increasing concentrations of MeJA from 0.004–4.45 μM with or without IAA and BA. The same concentrations of MeJA (0–4.45 μM) increased the cell growth with IAA and BA, when administered to the cultured cells in M9 medium. This was found to enhance the production of shikonin. The optimum time for MeJA addition for enhanced shikonin formation
was 4 d after cell inoculation in M9 medium. Furthermore, shikonin formation was affected significantly by both MeJA/IAA and MeJA/BA combinations. Shikonin content
was enhanced by increasing MeJA concentrations with IAA concentrations in the range of 0–28 μM and with BA concentrations in the range of 0–44.38 μM in MeJA/BA experiments, respectively. The optimal combination of MeJA and IAA was 4.45 μM and 0.28 μM, while MeJA and BA concentrations of 4.45 μM and 2.22 μM were optimal for shikonin formation. The result also showed that MeJA increased phenylalanine ammonia-lyase (PAL) and p-hydroxybenzoic acid-geranyltransferase (PHB-geranyltransferase) activites during the course of shikonin formation, but decreased
the activity of PHB-O-glucosyltransferase within 9 d after inoculation. These results suggest that enhanced shikonin formation in cultured Onosma paniculatum cells induced by MeJA involves regulation of the key enzyme activities. 相似文献
8.
Patrizzia Mastromatteo-Alberga Fabiola Placeres-Uray Marcelo A. Alfonzo-González Ramona Gonzalez de Alfonzo Itala Lippo de Becemberg 《Journal of receptor and signal transduction research》2016,36(3):278-287
Muscarinic antagonists, via muscarinic receptors increase the cAMP/cGMP levels at bovine tracheal smooth muscle (BTSM) through the inhibition of phosphodiesterases (PDEs), displaying a similar behavior of vinpocetine (a specific-PDE1 inhibitor). The presence of PDE1 hydrolyzing both cyclic nucleotides in BTSM strips was revealed. Moreover, a vinpocetine and muscarinic antagonists inhibited PDE1 located at plasma membranes (PM) fractions from BTSM showing such inhibition, an M2AChR pharmacological profile. Therefore, a novel Ca2+/CaM dependent and vinpocetine inhibited PDE1 was purified and characterized at PM fractions from BTSM. This PDE1 activity was removed from PM fractions using a hypotonic buffer and purified some 38 fold using two columns (Q-Sepharose and CaM-agarose). This PDE1 was stimulated by CaM and inhibited by vinpocetine showing two bands in PAGE-SDS (56, 58?kDa) being the 58?kDa identified as PDE1A by Western blotts. This PDE1A activity was assayed with [3H]cGMP and [3H]cAMP exhibiting a higher affinity as Km (μM) for cGMP than cAMP but being close values with Vmax cAMP/cGMP ratio of 1.5. The co-factor Mg2+ showed similar K(A) (mM) for both cyclic nucleotides. Vinpocetine showed similar inhibition concentration 50% (IC50 of 4.9 and 4.6?μM) for cAMP and cGMP, respectively. CaM stimulated the cyclic nucleotides hydrolysis by PDE1A exhibiting similar activation constant as K(CaM), in nM range. The original finding was the identification and purification of a vinpocetine and muscarinic antagonist-inhibited and CaM-activated PM-bound PDE1A, linked to M2AChR. A model of this novel signal transducing cascade for the regulation of cyclic nucleotides levels at BTSM is proposed. 相似文献
9.
Gerald Audesirk David Shugarts Gina Nelson Julie Przekwas 《In vitro cellular & developmental biology. Plant》1989,25(12):1121-1128
Summary Neurons from brains of chick embryos and pond snails (Lymnaea stagnalis) were cultured for 3 to 4 d in the presence of no toxins, inorganic lead (PbCl2), or organic lead (trielthyl lead chloride). In chick neurons, inorganic lead reduced the percentage of cells that grew neurites
(IC50=270μM total lead, approximately 70 nM free Pb2+) but did not reduce the number of neurites per cell or the mean neurite length. Triethyl lead reduced the percentage of cells
that grew neuites (IC50=0.24 μM) and the mean neurite length (extrapolated IC50=3.6 μM) but did not reduce the number of neurites per cell. InLymnaea neurons, inorganic lead reduced the percentage of cells that grew neurites (IC50=13 μM total lead; approximately 10 nM free Pb2+). Triethyl lead reduced the percentage of cells that grew neurites (IC50=0.4 μM) and exerted significant toxicity at 0.2 μM. The two forms of lead affected neurite growth in qualitatively different ways, which suggests that their mechansms of action
are different.
These experiments were supported by grants from the Environmental Protection Agency, Washington, DC, and the National Institutes
of Environmental Health Science, Research Triangle Park, NC. 相似文献
10.
Suleiman A. Suleiman Jeffrey B. Stevens 《In vitro cellular & developmental biology. Plant》1987,23(5):332-338
Summary Cell viability, cytochrome P-450 content, cell respiration, and lipid peroxidation were all investigated as a function of
oxygen tension in adult rat hepatocytes in short-term culture (less than 9 h). The various oxygen tensions used in this study
were obtained by equilibrating culture medium with air, air + nitrogen, or air + oxygen. Cell viability, as assessed by trypan
blue exclusion, was significantly greater at all time points tested when hepatocytes were cultured in Ham's F12 medium containing
132 μM O2, as compared to medium equilibrated with air (220 μM O2) or air + oxygen (298 μM O2). Cells cultured in 220 μM O2 (air) also exhibited a gradual loss of cytochrome P-450, so that by 9 h of incubation less than 60% of the active material
remained. This loss of P-450 was minimized when cells were cultured in 163 μM O2 and abolished when cells were cultured in 132 μM O2. The 132 μM O2 exposure conditions also maintained cell respiration at the 1 h incubation values, whereas there was a continuous loss in
cell respiration over time when the cells were cultured in either 220 μM O2 (air) or 298 μM O2 (air:O2). These cytotoxicity findings may be related to oxidative cell damage inasmuch as it was additionally demonstrated that lipid
peroxidation (as measured by malondieldehyde equivalents) was consistantly lower in hepatocytes cultured in air:N2 as compared to air or air:O2. These results suggest that hepatocyte culture in low oxygen tension improves not only cell viability but also maintains
other functional characteristics of the cell.
This work was supported by a Biomedical Research Support Grant S-S07-RR 05448 awarded to the University of Minnesota School
of Public Health by the Biomedical Research Grant Program, Division of Research and Resources, National Institutes of Health,
Bethesda, MD. 相似文献
11.
D. H. Maurice 《Cell biochemistry and biophysics》1998,29(1-2):35-47
Cultured rat aortic vascular smooth muscle cells (VSMC) express both cGMP- inhibited cAMP phosphodiesterase (PDE-3) and Ro,20-1724-inhibited cAMP phosphodiesterase (PDE-4) activities. Utilizing a PDE-3-selective inhibitor (cilostamide) and a PDE-4-selective inhibitor (Ro,20-1724), PDE-3 and PDE-4 activities were shown to account for 15 and 55% of total VSMC cAMP phosphodiesterase (PDE) activity. Incubations of VSMC with either forskolin or 8-bromo-cAMP caused a concentration- and time-dependent increase in total cellular cAMP PDE activity. In these cells, both PDE-3 and PDE-4 activities were increased, with a relatively larger effect observed on PDE-3 activity. Similar incubations with an activator of soluble guanylyl cyclase (sodium nitroprusside) or with 8-bromo-cGMP did not increase cAMP PDE activity. cAMP-induced increases in cAMP PDE activity were inhibited with actinomycin D or cycloheximide, demonstrating that new mRNA and protein synthesis were required. We conclude that VSMC cAMP PDE activity is elevated following long-term elevation of cAMP, and that increases in PDE-3 and PDE-4 activities account for more than 70% of this increase. These results may have implications for long-term use of cAMP PDE inhibitors as therapeutic agents. 相似文献
12.
Terry L. Riss' David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):136-142
Summary The effect of 17β-estradiol (E2) on growth of GH4C1 rat pituitary tumor cells was investigated under serum-free conditions and with medium containing charcoal-extracted serum.
Serum-free TRM-1 medium was a 1∶1 (vol/vol) mixture of F12-DME supplemented with 50 μg/ml gentamicin, 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 10 μg/ml insulin, 10 μg/ml transferrin, 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine, and 500 μg/ml bovine serum albumin. The cells grew continuously in TRM-1 but were E2 responsive only when growth was retarded by reducing the T3 concentration to 10 pM (TRM-MOD). Addition of 1 to 10 nM E2 to TRM-MOD increased growth by 0.3 to 0.9 cell population doublings over controls in 9 d. By using medium supplemented with
charcoal-extracted sera, basal growth became 1 to 1.5 cell population doublings in 9 d. Addition of 0.1 pM E2 to medium containing charcoal-extracted serum caused a significant increase in cell number whereas pM-nM concentrations stimulated 200 to 570% increases over controls. The effect of steroid hormone was the same in phenol-red-containing
and indicator-free medium. The data presented confirm that the major requirements for demonstration of estrogenic effects
in culture were optimum concentrations of thyroid hormones and the presence of yet-to-be-characterized serum factors.
This work was supported by National Cancer Institute (Bethesda, MD) grants CA-26617 and CA-38024, American Cancer Society
grant BC-255, and grant 2225 from The Council for Tobacco Research, U.S.A., Inc. 相似文献
13.
Mitsuru Iwata Shoko Iwata Mark A. Everett Bryan B. Fuller 《In vitro cellular & developmental biology. Plant》1990,26(6):554-560
Summary A human foreskin organ culture system has been developed to study the response of human skin to hormonal stimulation. Foreskins
are maintained in culture on floating plastic supports which allows the epidermal surface to be exposed to air while the dermis
is bathed in nutrient medium. Both black and white human foreskins can be maintained in organ culture for at least 1 wk with
no change in the tissue structure or cell viability as determined by histochemical staining and by dopa reaction staining.
Tyrosinase activity in both black and white human foreskin cultures decays markedly during the first 2 d of culture to a new
steady state level which remains stable throughout the culture period. Both black and white foreskin cultures consistently
demonstrate 2- to 10-fold increases in tyrosinase activity when treated with theophylline (1 mM). Approximately 90% of all skin cultures examined showed an increase in enzyme activity when treated with this phosphodiesterase
inhibitor. Dibutyryl cAMP (0.1 mM) and [Nle4, D-phe7]-alpha MSH (10−8
M), were also found to markedly stimulate tyrosinase activity in some skin cultures, whereas alpha-MSH and prostaglandin E1 produced only an inconsistent and small increase in the activity of the enzyme. Histamine (1 μM), vitamin D3 (1 μM), and retinoic acid (1μM) failed to stimulate tyrosinase activity in either white or black foreskin cultures. This hormone-responsive organ culture
system can be utilized to characterize the molecular processes responsible for the regulation of tyrosinase and pigmentation
in human skin.
This work was supported by a research contract from the Oklahoma Center for the Advancement of Science and Technology (OCAST)
and by a research grant from the Presbyterian Health Foundation. 相似文献
14.
Summary Skeletal muscle hypertrophy is promoted in vivo by administration of β-adrenergic receptor (βAR) agonists. Chicken skeletal
muscle cells were treated with 1 μM isoproterenol, a strong βAR agonist, between days 7 and 10 in culture. βAR population increased by approximately 40% during
this treatment; however, the ability of the cells to synthesize cyclic adenosine monophosphate (cAMP) was diminished by twofold.
Neither the basal concentration of cAMP nor the quantity of myosin heavy chain (MHC) was affected by the 3-d exposure to isoproterenol.
To understand further the relationship between intracellular cAMP levels, βAR population, and muscle protein accumulation,
intracellular cAMP levels were artificially elevated by treatment with 0–10 μM forskolin for 3 d. The basal concentration of cAMP in forskolintreated cells increased up to sevenfold in a dose-dependent
manner. Increasing concentrations of forskolin also led to an increase in βAR population, with a maximum increase of approximately
40–60% at 10 μM forskolin. A maximum increase of 40–50% in the quantity of MHC was observed at 0.2 μM forskolin, but higher concentrations of forskolin reduced the quanity of MHC back to control levels. At 0.2 μM forskolin, intracellular levels of cAMP were higher by approximately 35%, and the βAR population was higher by approximately
30%. Neither the number of muscle nuclei fused into myotubes nor the percentage of nuclei in myotubes was affected by forskolin
at any of the concentrations studied. 相似文献
15.
Terry L. Riss Betty H. Stewart David A. Sirbasku 《In vitro cellular & developmental biology. Plant》1989,25(2):127-135
Summary The growth of GH4C1, GH3, GH1, and GH3C15 rat pituitary tumor cell lines was studied in a serum-free medium (designated TRM-1) formulated with 1∶1 (vol/vol) mixture
of Ham's F12 nutrient mixture and Dulbecco's modified Eagle's medium (F12-DME) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 50 μg/ml gentamicin supplemented with 10 μg/ml bovine insulin,
10 μg/ml human transferrin (Tf), 10 ng/ml selenous acid, 10 nM 3,5,3′-triiodothyronine (T3), 50 μM ethanolamine (Etn), and 500 μg/ml bovine serum albumin. Of the lines evaluated, only the GH1 failed to grow in TRM-1. Passage of the GH4C1 and GH3 lines from serum-containing medium into TRM-1 caused an initial selection resulting in cells that grew progressively at higher
rates and finally were maintained indefinitely in TRM-1. These populations showed a requirement for supraphysiologic concentrations
of T3 (1.0 to 10 nM). After adaptation of the GH4C1 line in TRM-1 for ≤20 generations, removal of components gave a less complex mixture containing 15 mM HEPES, 50 μ/ml gentamicin, 10 μg/ml Tf, 10 nM T3, and 50 μM Etn (designated TRM-2) that supported serial passage of the cells. Under these conditions, thyroid hormone dependence was
lost progressively. When T3 was removed from TRM-2 adapted cells, a third population was selected that no longer required thyroid hormones and was only
slightly stimulated by T3. These studies demonstrated that the combination of serum-containing and serum-free conditions can be used to select pituitary
cell populations that a) required both serum-factor(s) and T3 for optimum growth, b) required supraphysiologic concentrations of T3 without serum proteins other than Tf and albumin, and c) were completely autonomous in that they proliferated in medium supplemented
only with Tf and nutrients without necessity of other serum factor(s) or T3.
This work was supported by grants CA-26617 and CA-38024 from the National Cancer Institute, Bethesda, MD, American Cancer
Society grant BC-255, and grant 2225 from the Council for Tobacco Research, Inc., USA. 相似文献
16.
Susan Mukavitz Kramer Ursula E. M. Gibson Brian M. Fendly Marjorie A. Mohler Daniel W. Drolet Paul D. Johnston 《In vitro cellular & developmental biology. Plant》1990,26(6):647-656
Summary A novel relaxin sensitive cell line of apparent smooth muscle origin has been established from a newborn rhesus monkey uterus
(NRMU). NRMU cells respond to relaxin, in the presence of 1 μM forskolin, by producing intracellular adenosine 3′, 5′-cyclic monophosphate (cAMP). The increase in cAMP levels is dose,
time and cell density dependent, reaching peak levels at 10 min when cells are seeded at 1×105 cells/well. Specificity was demonstrated by neutralization of the relaxin activity with anti-relaxin monoclonal and polyclonal
antibodies, degradation of cAMP in the presence of phosphodiesterase, and confirmation of the absence of cGMP. Three synthetic
analogs of human relaxin generated a dose-related cAMP response as did synthetic native human relaxin. Natural relaxin purified
from human corpora lutea tissue also generated a response similar to synthetic human relaxin. Porcine and rat relaxins also
increased levels of cAMP. Insulin, but not IGF I or IGF II, was capable of increasing cAMP levels in NRMU cells, however,
200 ng/mL were required to achieve cAMP levels comparable to 6.25 ng/ml relaxin. Combinations of relaxin with insulin, IGF
I or IGF II did not increase cAMP levels above levels obtained with relaxin alone. The effect on NRMU cells of other hormones,
growth factors and drugs potentially present in cell culture systems or serum samples was evaluated. In combination with relaxin,
oxytocin significantly decreased the cAMP production below the levels induced by relaxin alone, whereas progesterone and prostaglandin
E2 resulted in additive increases in cAMP. These data suggest that the NRMU cell line is an appropriate target tissue for studying
relaxin-mediated biological responsesin vitro as well as functioning as the primary component of a relaxinin vitro bioassay.
Editor's statement This paper details a smooth muscle cell line that is responsive to relaxin and provides a useful assay
system for the hormone, as well as providing a model system for the study of the mechanisms of relaxin action. 相似文献
17.
Ellen Borenfreund Harvey Babich Nieves Martin-Alguacil 《In vitro cellular & developmental biology. Plant》1990,26(11):1030-1034
Summary Neutral red assay, as an index of cytotoxicity, has been applied to predictive screening of chemotherapeutic agents. Human
hepatoma and melanoma tumor cells and normal melanocytes, keratinocytes and fibroblasts were incubated for 2, 24, and 48 h
with graded concentrations of cis-platinum (0.1 to 80 μM), doxorubicin (0.01 to 100 μM), and 5-fluorouracil (1 to 1000 μM). Cells were most sensitive after 48 h. Tumor cells, based on 50% toxicity values, were 2–4 times more sensitive than the
normal cells, except for cis-platinum, where only melanoma cells, as compared to normal melanocytes, showed a marked difference
in cytotoxic response. Methotrexate (1 to 10 μM) toxicity could be reversed in the presence of 100 μM of leucovorin. This sensitive, rapid, and economical assay is suitable for preclinical screening and drug development.
This work has been supported, in part, by funds from Schering Corporation, New Jersey, and Chevron Environmental Health Center,
Inc., California. 相似文献
18.
Amanda G. Vang Shlomo Z. Ben-Sasson Hongli Dong Barbara Kream Michael P. DeNinno Michelle M. Claffey William Housley Robert B. Clark Paul M. Epstein Stefan Brocke 《PloS one》2010,5(8)
Background
Abolishing the inhibitory signal of intracellular cAMP by phosphodiesterases (PDEs) is a prerequisite for effector T (Teff) cell function. While PDE4 plays a prominent role, its control of cAMP levels in Teff cells is not exclusive. T cell activation has been shown to induce PDE8, a PDE isoform with 40- to 100-fold greater affinity for cAMP than PDE4. Thus, we postulated that PDE8 is an important regulator of Teff cell functions.Methodology/Principal Findings
We found that Teff cells express PDE8 in vivo. Inhibition of PDE8 by the PDE inhibitor dipyridamole (DP) activates cAMP signaling and suppresses two major integrins involved in Teff cell adhesion. Accordingly, DP as well as the novel PDE8-selective inhibitor PF-4957325-00 suppress firm attachment of Teff cells to endothelial cells. Analysis of downstream signaling shows that DP suppresses proliferation and cytokine expression of Teff cells from Crem −/− mice lacking the inducible cAMP early repressor (ICER). Importantly, endothelial cells also express PDE8. DP treatment decreases vascular adhesion molecule and chemokine expression, while upregulating the tight junction molecule claudin-5. In vivo, DP reduces CXCL12 gene expression as determined by in situ probing of the mouse microvasculature by cell-selective laser-capture microdissection.Conclusion/Significance
Collectively, our data identify PDE8 as a novel target for suppression of Teff cell functions, including adhesion to endothelial cells. 相似文献19.
Summary
In vitro methods were applied to the only remaining plant of the Meelup Mallee (Eucalyptus phylacis), a critically endangered species from the southwest of Western Australia. Shoot explants were initiated into culture using
a 1/2 MS [Murashige and Skoog basal medium (BM) for all experiments] liquid medium supplemented with 1% (w/v) activated charcoal,
which was replenished twice daily, followed by transfer of explants to agar medium supplemented with 0.5 μM zeatin. Explants were cultured under low intensity lighting (PPFD of 5–10 μmol m−2s−1) to minimize blackening of tissues, and some explants were induced to produce nodular green calluses in response to BM supplemented
with 5 μM thidiazuron. Nodular green calluses were induced to form adventitious shoots following transfer to medium supplemented with
0.5 μM zeatin and 1 μM gibberellic acid, A4 isomer (GA4). Development of shoots was completed on 1 μM zeatin + 0.1 μM 6-benzylaminopurine (BA) in vented culture tubes. Regenerated shoots were sequentially cultured on medium containing 0.5
μM zeatin + 0.2 μM indoleacetic acid (IAA) followed by either 0.5 μM zeatin + 1μM GA4 for shoot elongation or 1 μM zeatin + 0.5 μM IAA to optimize shoot growth. Rooted microshoots were produced after 4 weeks on 5 μM indolebutyric acid (IBA) and survived acclimatization and transfer to potting mixture. 相似文献
20.
Larentis AL Almeida RV Rössle SC Cardoso AM Almeida WI Bisch PM Alves TL Martins OB 《Cell biochemistry and biophysics》2006,44(3):530-538
The enzyme 2′-aminobiphenyl-2,3-diol-1,2-dioxygenase (CarB), encoded by two genes (carBa and carBb), is an α2β2 heterotetramer that presents meta-cleavage activity toward the hydroxylated aromatic ring in the carbazole degradation pathway from petroleum-degrader bacteria
Pseudomonas spp. The 1082-base, pair polymerase chain reaction product corresponding to, carBaBb genes from Pseudomonas stutzeri ATCC 31258 was cloned by site-specific recombination and expressed in high levels in Escherichia coli BL21-SI with a histidine-tag and in native form. The CarB activity toward 2,3-dihydroxybiphenyl was similar for these two
constructions. The α2β2 3D model of CarB dioxygenase was proposed by homology modeling using the protocatechuate 4,5-dioxygenase (LigAB) structure
as template. Accordingly, His12, His53, and Glu230 coordinate the Fe(II) in the catalytic site at the subunit CarBb. The model
also indicates that His182 is the catalytic base responsible for deprotonating one of the hydroxyl group of the substrate
by a hydrogen bond. The hydrophobic residues Trp257 and Phe258 in the CarB structure substituted the LigAB amino acid residues
Ser269 and Asn270. These data could explain why the CarB was active for 2,3-dihydroxybiphenyl and not for protocatechuate. 相似文献