Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The effect of 6-dimethylaminopurine (6-DMAP) on germinal vesicle breakdown (GVBD) and maturation in bovine oocytes was investigated in this study. This puromycin analog has been shown to be an inhibitor of phosphorylation. Whereas GVBD occurred in nearly all oocytes (96.8%, 120/124) in control medium, presence of 6-DMAP (2 mM) blocked this process almost completely, irrespective of the presence (98.3% GV, 349/355) or absence (97.1% GV, 165/170) of cumulus cells. When lower concentrations of 6-DMAP were used (100-500 microM), GVBD was observed in 87.9% of oocytes, but their maturation was arrested at late diakinesis-metaphase I stage. The inhibition of GVBD was fully reversible, but most of the metaphase II plates were abnormal (80%). To assess whether the action of 6-DMAP is different from the inhibitors of protein synthesis, metaphase II oocytes were exposed to either cycloheximide or 6-DMAP, respectively. Whereas in cycloheximide-supplemented medium approximately 80% of the oocytes were activated, parthenogenetic activation was much less frequent after incubation in 6-DMAP (14.5%). Fusion studies showed that, even if GVBD occurs in 6-DMAP supplemented medium, the level of the maturation-promoting factor (MPF) is decreased. These experiments may indicate the importance of phosphorylation for GVBD in cattle oocytes.
Mol Reprod Dev 1991 Aug
PMID:Effect of 6-dimethylaminopurine on germinal vesicle breakdown of bovine oocytes. 188 17

In denuded mouse oocytes, neither 3 nor 5 hours of preincubation in dbcAMP (1 mM) and cycloheximide (10 micrograms/ml), followed by further 3 hours in cycloheximide only, lowered the rate of GVBD (93% and 92%, respectively). It means that 3 and 5 hours preincubation in cycloheximide did not impair the ability of mouse oocytes to resume meiosis in medium with the protein synthesis inhibitor. To test the combined effects of inhibition of protein phosphorylation and protein synthesis, oocytes were cultured for 3, 4, or 5 hours in 2 mM of 6-DMAP and subsequently for 3 hours in 10 micrograms/ml cycloheximide. The incubation in 6-DAMP for 4 or 5 hours diminished (63% or 35% of GVBD, respectively) the ability of mouse oocytes to resume meiosis when subsequent protein synthesis was blocked by cycloheximide. However, the highly condensed bivalents were always visible in GVs. Thus the above treatment did not prevent chromatin condensation although GVBD was blocked.
Mol Reprod Dev 1990 Nov
PMID:Combined effects of protein synthesis and phosphorylation inhibitors on maturation of mouse oocytes in vitro. 196 58

The amount and distribution of glial fibrillary acidic protein (GFAP) were determined by flow cytometry (FCM) and an enzyme-linked immunosorbent assay (ELISA) in five human glioma cell lines stained by the indirect immunofluorescence method using anti-GFAP monoclonal antibody. Standard reference beads containing a known amount of fluorescein were used to calibrate the flow cytometer; however, the intensity of fluorescence from these beads was too weak to allow direct comparison with the fluorescence from the stained cells. Therefore, the flow cytometer was recalibrated using reference beads with a fluorescence intensity similar to that of the glioma cells. By comparing the fluorescence intensities of the two types of reference beads, it was possible to determine the fluorescein content of the stained cells directly from the relative fluorescence intensity (channel number). Glioma cell lines 343 MGA, SF 126, SF 188, U 251, and U 87 had fluorescein concentrations of 72.0 +/- 6.8, 8.1 +/- 0.3, 52.6 +/- 3.1, 86.4 +/- 4.0, and 56.2 +/- 2.9 x 10(5) (mean +/- standard error) Eq Sol Mol (equivalent solution of mole), respectively. The GFAP content of these cell lines, determined by ELISA, was 15.7 +/- 5.2, 0.5 +/- 0.1, 11.1 +/- 2.0, 20.8 +/- 4.6, and 9.5 +/- 2.7 pg GFAP/cell, respectively, and correlated closely with the results of FCM (R = 0.983, p less than 0.0028). A linear regression analysis yielded the following equation: pg GFAP/cell = -2.3376 + 0.2518 x FCM integrated mean channel number (fluorescein concentration: 10(5) Eq Sol Mol).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Quantitation and distribution analysis of glial fibrillary acidic protein in human glioma cells in culture. 276 8

Silencing of Nia host genes and transgenes (encoding nitrate reductase) was previously achieved by introducing into tobacco plants the tobacco Nia2 cDNA cloned downstream of the cauliflower mosaic virus (CaMV) 35S promoter. To check whether Nii host genes and transgenes (encoding nitrite reductase, the second enzyme of the nitrate assimilation pathway) were also susceptible to silencing, a transgene consisting of the tobacco Nii1 gene with two copies of the enhancer of the 35S promoter cloned 1 kb upstream of the Nii promoter region was introduced into tobacco plants. Among nine independent transformants analysed, two showed silencing of Nii host genes and transgenes in some descendants after selfing, but never after back-crossing with wild-type plants, suggesting that silencing depends on the number of transgene loci and/or on certain allelic or ectopic combinations of transgene loci. In one transformant carrying a single transgene locus in a homozygous state, silencing was triggered in all progeny plants of each generation, 20 to 50 days after germination. Field trial analysis confirmed that silencing was not triggered when the transgene locus of this latter line was present in a hemizygous state. In addition, it was revealed that silencing can be triggered, albeit at low frequency and later during the development, when this transgene locus is brought into the presence of a non-allelic transgene locus by crossing, suggesting that a homozygous state is not absolutely required.
Mol Gen Genet 1995 Aug 21
PMID:Molecular and genetic analysis of nitrite reductase co-suppression in transgenic tobacco plants. 756 93

Effects of phorbol myristate acetate (PMA), dibutyryl cyclic AMP (dbcAMP), 6-dimethylaminopurine (6-DMAP), and okadaic acid (OA) on plasminogen activator (PA) activity in porcine oocyte-cumulus cell complexes (POCC) in vitro were determined. Cumulus cell-enclosed oocytes were collected from 1-4 mm antral follicles and cultured in TCM-199 with 0.3% polyvinylpyrrolidone for 48 hr. PA activities in POCC were quantified using SDS-PAGE, casein-agar zymography, and densitometry. Two plasminogen-dependent lytic zones (93-96 kD and 71-79 kD) were observed in POCC. Addition of amiloride to the zymography, a competitive inhibitor of urokinase-type PA, failed to reduce activities in either zone, suggesting that the 71-79 kD band is a tissue-type PA (tPA) and the 93-96 kD band is possibly a tPA-inhibitor complex. Changes in PA activity due to the various treatments were expressed relative to the PA activity in 40 POCC. Increasing dbcAMP increased PA (P < 0.05) activity in dose-dependent fashion, whereas 6-DMAP and 10 and 100 ng/ml PMA inhibited (P < 0.05) PA activity. PA activity increased (P < 0.05) in POCC treated with up to 25 nM OA; however, activity decreased (P < 0.05) at concentrations > 75 nM. Treatment with 25 nM OA also induced the expression of an amiloride-sensitive PA (49-52 kD). Germinal vesicle breakdown and progression to metaphase II were inhibited (P < 0.05) by 2.5 mM dbcAMP and 2 mM 6-DMAP, whereas 100 ng/ml PMA and 25 nM OA inhibited (P < 0.05) only progression to metaphase II.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1995 Mar
PMID:Effects of stimulators of protein kinases A and C and modulators of phosphorylation on plasminogen activator activity in porcine oocyte-cumulus cell complexes during in vitro maturation. 777 47

One approach towards understanding the transduction pathways of phytochromes is the selection of mutants impaired in various steps. We report here the construction of an inducible counter-selection system for such mutants employing the enzyme nitrate reductase. This enzyme can convert the benign substrate analogue chlorate to the toxic product chlorite, resulting in severe growth inhibition. An Arabidopsis thaliana nitrate reductase gene (Nial*2) was placed under the regulation of an Arabidopsis thaliana light-harvesting chlorophyll a/b protein (Lhcb1*3) promoter that is phytochrome-responsive. The chimeric Lhcb::Nia gene was transformed into A. thaliana. Homozygous transformant lines were selected and grown in the absence of nitrate and the presence of L-glutamine, conditions that substantially inhibited the expression of the endogenous nitrate reductase genes. In darkness seedlings of the transformed lines were resistant to chlorate; however, when seedlings were grown with intermittent red light, increased sensitivity to chlorate was observed. This sensitivity was correlated with an increase in both Nia1*2 RNA levels and nitrate reductase activity. The resistant seedlings were clearly distinguishable from the sensitive ones based on hypocotyl length, with no overlap in this parameter between the two populations. Thus, this system should allow for the selection of mutants that are impaired in phytochrome regulation of the transcription of Lhcb genes.
Plant Mol Biol 1995 Jan
PMID:A chimeric Lhcb::Nia gene: an inducible counter selection system for mutants in the phytochrome signal transduction pathway. 786 82

Protein phosphorylation patterns were studied by radiolabelling goat cumulus oocyte complexes with [32P]orthophosphate for various periods of time. The radiolabelled denuded oocytes were assessed for nuclear status and were used individually for gel electrophoresis. This study demonstrated that specific changes in protein phosphorylations were programmed during goat oocyte maturation. One of the most prominent changes was a general increase in the phosphorylation rate at germinal vesicle breakdown (GVBD). From 8 hr of culture, dominant phosphoprotein bands with apparent molecular weights of 27, 31, 40, and 50 kD were observed; they remained at this level until the metaphase II stage. In the molecular weight range of 65-80 kD, the protein phosphorylation pattern exhibited characteristic differences, with a complex series of phosphoproteins appearing and disappearing, duration maturation. Addition of 6-dimethylaminopurine (6-DMAP) at the onset of culture blocked the maturation process after GVBD and induced a dramatic condensation of chromatin. When added at different times after GVBD, 6-DMAP invariably induced chromosome condensation. This inhibition was partly reversible; i.e., after removal of the drug, oocytes were able to progress only until metaphase I.
Mol Reprod Dev 1993 Dec
PMID:Protein phosphorylation patterns during in vitro maturation of the goat oocyte. 830 14

Phosphorylation is considered as a common post-translational modification implicated in the control of various key enzymes. In somatic and germinal cells, important regulators of the cell cycle are controlled by their phosphorylation status, and some act as kinases or phosphatases themselves. Bovine oocytes are blocked in the germinal vesicle (GV) stage until either an LH surge occurs or until oocytes are released from the inhibitory influence of the follicle. Meiotic resumption in vitro is therefore an excellent model for the study of phosphorylation events that occur in the G2/M transition, a control point of the cellular cycle. To better understand this transition, we have modulated, either directly or indirectly, kinases using known effectors (epidermal growth factor, EGF; isobutylmethylxanthine-forskolin, Bx-Fk; 6-dimethylaminopurine, 6-DMAP) or phosphatases (okadaic acid, OA) or cycloheximide, which is known to inhibit maturation through protein synthesis suppression. With this procedure, influence on meiotic resumption and phosphoprotein patterns was verified. Both EGF and OA accelerated nuclear maturation after 9 hr of culture. Only 23% (n = 140) and 9% (n = 111) of oocytes were still at GV stage with EGF and OA, respectively, compared to 41% (n = 105) of control oocytes. The different treatments changed the protein patterns in oocytes. In cumulus cells, the patterns were especially modified by the OA treatment. Characteristic changes that occur in germ cells were also identified. Nuclear maturation was inhibited by modulators of kinase (6-DMAP, GV = 74%, n = 126; cAMP dependent protein kinase (PKA) stimulators, Bx-Fk, GV = 71%, n = 129) likewise, phosphoprotein patterns were affected, especially in oocytes.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1995 Sep
PMID:Effects of different kinases and phosphatases on nuclear and cytoplasmic maturation of bovine oocytes. 856 45

The interaction of microtubule associated proteins (MAPs) with the microtubule system has been characterized in depth in neuronal cells from various mammalian species. These proteins interact with well-defined domains within the acidic tubulin carboxyl-terminal regulatory region. However, there is little information on the mechanisms of MAPs-tubulin interactions in nonmammalian systems. Recently, a novel tau-like protein designated as DMAP-85 has been identified in Drosophila melanogaster, and the regulation of its interactions with cytoskeletal elements was analyzed throughout different developmental stages of this organism. In this report, the topographic domains involved in the binding of DMAP-85 with tubulin heterodimer were investigated. Affinity chromatography of DMAP-85 in matrixes of taxol-stabilized microtubules showed the reversible interaction of DMAP-85 with domains on the microtubular surface. Co-sedimentation studies using the subtilisin-treated tubulin (S-tubulin) indicated the lack of association of DMAP-85 to this tubulin moiety. Moreover, studies on affinity chromatography of the purified 4 kDa C-terminal tubulin peptide bound to an affinity column, confirmed that DMAP-85 interacts directly with this regulatory domain on tubulin subunits. Further studies on sequential affinity chromatography using a calmodulin affinity column followed by the microtubule column confirmed the similarities in the interaction behaviour of DMAP-85 with that of tau. DMAP-85 associated to both calmodulin and the microtubular polymer. These studies support the idea that the carboxyl-terminal region on tubulin constitutes a common binding domain for most microtubule-interacting proteins.
Mol Cell Biochem 1996 May 24
PMID:Tubulin domains for the interaction of microtubule associated protein DMAP-85 from Drosophila melanogaster. 881 77

6-dimethylaminopurine (6-DMAP), a potent protein kinase inhibitor, drives most cells into an interphasic stage. Experiments were undertaken with oocytes from three marine invertebrate species, i.e., Mytilus edulis, Spisula solidissima, and Strongylocentrotus droebachiensis, wherein oocytes were arrested at different phases of meiosis. 6-DMAP induced a continuous DNA synthesis in meiotic cells, whereas it allowed a single round of DNA replication in treated mitotic cells, regardless of species considered. The effects of 6-DMAP were accompanied in all cases by rephosphorylation on tyrosine of the p34cdc2 homolog, the M-phase promoting factor (MPF) catalytic subunit. The fact that 6-DMAP overcomes the inhibitory control of replication during meiosis suggests that this process depends upon protein phosphorylation, while DNA synthesis regulation in mitotic cells relies on 6-DMAP-insensitive events.
Mol Reprod Dev 1996 Aug
PMID:DNA replication initiation by 6-DMAP treatment in maturing oocytes and dividing embryos from marine invertebrates. 884 86


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