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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In this report we introduce a simple, fast, and reliable method to prepare whole cell or nuclear extracts from small numbers of cells. These extracts were used to study transcriptional activation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) in vitro. Our results revealed that the time courses of activation of extracts derived from cells stimulated with the mitogenic lectin phytohemagglutinin (PHA) or with the tumor promoter phorbol 12-myristate 13-acetate (PMA) are different. PMA induces a rapid onset of increased in vitro transcription from the HIV-1 LTR, while PHA causes a slow and sustained response. The biochemical relevance of protein synthesis inhibition by cycloheximide treatment of cells was investigated. In these studies, PMA induction of a change in in vitro transcriptional activity is not dependent on protein synthesis. Cycloheximide alone is insufficient to induce activation. Oligonucleotide-mediated site-directed mutagenesis demonstrated that mutation of the TATA box in the LTR ablated initiation of both basal-level transcription and activation by extracts from cells stimulated with PMA. Surprisingly, mutation of both kappa B sites in the LTR reduced but did not eliminate the in vitro response to extracts prepared at early time points after PHA or PMA stimulation of Jurkat cells. The reduction was greater in extracts derived from cells treated with PMA. Deletion analysis of the HIV-1 LTR revealed at least one region (-464 to -252) capable of suppressing in vitro transcription in extracts from Jurkat cells stimulated by PMA. This result is consistent with early studies of the HIV-1 LTR in transient transfection assays. We therefore have been able to observe distinct regulatory events at early time points after cells are exposed to agents known to induce transcription of both the HIV-1 LTR reporter gene constructs and the HIV-1 provirus itself.
Mol Cell Biol 1991 Apr
PMID:An in vitro transcription analysis of early responses of the human immunodeficiency virus type 1 long terminal repeat to different transcriptional activators. 200 86

HTC rat hepatoma cells synthesize and secrete tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor type 1 (PAI-1). Incubation with 8-bromo-cAMP increases tPA activity more than 50-fold and, in combination with dexamethasone, causes an additional 4-fold increase. We have investigated the mechanism of the regulation of tPA activity by cyclic nucleotides, both alone and in combination with dexamethasone, by examining the effects of these agents on tPA and PAI-1 mRNA and protein. 8-Bromo-cAMP induces only a 2-fold increase in tPA mRNA and a 5-fold increase in tPA protein which is not sufficient to account for the increase in tPA activity. However, 8-bromo-cAMP causes a 90% decrease in PAI-1 mRNA and a 60-70% decrease in PAI-1 protein, which, taken together with the modest increase in tPA mRNA and protein, can account for the increase in tPA activity. Incubation with 8-bromo-cAMP plus dexamethasone also results in an 80-90% decrease in PAI-1 mRNA, but causes a synergistic 10- to 20-fold increase in tPA mRNA and protein. Regulation of both mRNAs by 8-bromo-cAMP requires concomitant RNA synthesis. Inhibition of protein synthesis by cycloheximide totally blocks the 8-bromo-cAMP-induced decrease in PAI-1 mRNA. Cycloheximide alone causes a 5- to 10-fold increase in tPA mRNA, and no further hormonal effect is observed. Thus, 8-bromo-cAMP increases tPA activity primarily by decreasing PAI-1 mRNA accumulation.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1990 Jan
PMID:Cyclic nucleotide regulation of plasminogen activator and plasminogen activator-inhibitor messenger RNAs in rat hepatoma cells. 215 75

To determine whether hypoxia has a direct effect on the plasma membrane transport of serotonin (5-HT), we measured 5-HT transport activity: (1) in plasma membrane vesicles isolated from normoxic and hypoxic endothelial cells, (2) in endothelial cell plasma membrane vesicles that were exposed directly to normoxia or hypoxia, and (3) in endothelial cell monolayers incubated in the presence of 1 x 10(-7) M cycloheximide and exposed to normoxia or hypoxia. A 24-h exposure of endothelial cells to hypoxia resulted in a 40% increase (P less than 0.005) in specific 5-HT transport by plasma membrane vesicles derived from these cells. When plasma membrane vesicles were isolated and then directly exposed to normoxia or hypoxia for 1 h at 37 degrees C, a 31% increase (P less than 0.005) in specific 5-HT transport was observed in hypoxic vesicles. Hypoxia did not alter the Km of 5-HT transport (normoxia = 3.47 microM versus hypoxia = 3.76 microM) but markedly increased the maximal rate of transport (Vmax) (normoxia = 202.4 pmol/min/mg protein versus hypoxia = 317.9 pmol/min/mg protein). Cycloheximide alone had no effect on 5-HT transport by normoxic endothelial cells but did block hypoxia-induced increases in 5-HT uptake in endothelial cell monolayers exposed to 24-h hypoxia. These results indicate that hypoxia increases 5-HT transport in pulmonary artery endothelial cells by a direct effect on the plasma membrane, leading to an increase in the effective number of transporter molecules without alteration in transporter affinity for 5-HT, and possibly by an indirect effect involving de novo protein synthesis.
Am J Respir Cell Mol Biol 1990 Oct
PMID:Hypoxia directly increases serotonin transport by porcine pulmonary artery endothelial cell plasma membrane vesicles. 220 39

The number of nuclear and cytosolic estrogen receptors (ER) per cell and the steady-state levels of the mRNA encoding a tissue-specific, estrogen-inducible protein (FOSP-1) were measured as a function of time following the addition of estradiol-17 beta (E2) to primary cultures of Xenopus oviduct cells. After a lag period of about 12 h, 10(-9) to 10(-7) M E2 caused a 10 to 15-fold increase in FOSP-1 mRNA by 60 h, whereas it was only 2-fold with 10(-7) M progesterone. Under the same conditions, E2 doubled its own total receptor content within the first 12 h, reaching a 4-fold increase in nuclear ER by 48 h. Cycloheximide treatment in the presence of 10(-7) M estradiol reduced the functional ER content by 75.90%. Treatment with the anti-estrogen ICI 164,384 of oviduct cells in which FOSP-1 mRNA was pre-induced to high levels with the hormones caused a drastic reduction in nuclear ER and a total loss of FOSP-1 mRNA in 72 h. The close correlation between the kinetics of autoinduction of ER and the induction of FOSP-1 mRNA, as was shown earlier for vitellogenin mRNA in hepatocytes (Perlman et al. (1984) Mol. Cell. Endocrinol. 38, 151-161), strongly suggests that Xenopus egg protein gene activation by estrogen requires the up-regulation of its own receptor by the hormone.
Mol Cell Endocrinol 1990 Jul 09
PMID:Autoinduction of estrogen receptor is associated with FOSP-1 mRNA induction by estrogen in primary cultures of Xenopus oviduct cells. 221 28

The rat prolactin gene is expressed at a high basal level in the pituitary tumor GH3 cell line. Culturing GH3 cells in a low-Ca2+, serum-free medium (SFM) depresses prolactin mRNA levels, and subsequent addition of Ca2+ to the SFM results in a specific, gradual, and sustained increase in prolactin mRNA levels. We have now examined whether the observed increase in prolactin mRNA levels can be attributed solely to an increase in the transcriptional rate of the prolactin gene. Treatment of GH3 cells in SFM with 0.4 mM CaCl2 for 24 to 48 h increased cytoplasmic prolactin mRNA levels by 5- to 10-fold, whereas the transcriptional rate of the prolactin gene was increased by less than twofold over values for SFM controls. Prolactin mRNA levels increased progressively during the 24-h period after Ca2+ addition, whereas prolactin gene transcription never exceeded a twofold increase over values for SFM controls. The activities of nuclear extracts from control and Ca2(+)-induced cells were examined in an in vitro transcription assay. The two extracts directed transcription from the prolactin promoter and the adenovirus major late promoter equally well. Cycloheximide had no effect on the ability of Ca2+ to increase or maintain prolactin mRNA levels. In dactinomycin mRNA clearance experiments, prolactin mRNA was cleared at the same rate in the absence and presence of Ca2+. These results demonstrate that although Ca2+ has a small effect on the transcriptional rate of the prolactin gene, Ca2+ produces a significant increase in prolactin mRNA levels by acting at a posttranscriptional site(s). Furthermore, Ca(2+) appears to increase prolactin mRNA levels by posttranslational modification of a stable protein, probably at a nuclear site.
Mol Cell Biol 1990 Feb
PMID:Transcriptional and posttranscriptional regulation of the rat prolactin gene by calcium. 230 47

Basic fibroblast growth factor (bFGF) is a potent autocrine and paracrine mitogen for cells of mesodermal origin. Although the protein is present in substantial amounts in a variety of tissues, the level of mRNA is undetectable in most normal tissues. This has led to speculation that bFGF mRNA is very unstable, but the half-life of this mRNA has not been described. A number of mRNAs encoding growth factors and growth-related proteins are known to be short-lived and posttranscriptionally regulated. In the present study we have examined the half-life of bFGF mRNA in two human tumor cell lines, which contain high (U87-MG) and low (T98-G) steady state bFGF mRNA levels. The half-life of bFGF mRNA, determined after transcriptional arrest with actinomycin-D, was approximately 10 min in T98-G cells, but was extended to 120 min in the presence of cycloheximide. In contrast, bFGF transcripts in U87-MG cells were very stable with a half-life considerably greater than 5 h. This was not attributable to a general stabilization of mRNA in the U87-MG line, since the half-life of c-myc mRNA in the two cell lines was similar (10 and 15 min in T98-G and U87-MG, respectively). Cycloheximide had no effect on the steady state level of bFGF in U87-MG cells. These findings suggest that posttranscriptional processes play an important role in the regulation of bFGF transcript levels and demonstrate that loss of posttranscriptional regulation could contribute to elevated bFGF expression in some tumors.
Mol Endocrinol 1990 Feb
PMID:Messenger RNA stabilization accounts for elevated basic fibroblast growth factor transcript levels in a human astrocytoma cell line. 232 99

A novel form of regulation of expression of a vertebrate heat shock gene is described. A cDNA clone encoding human Hsp27 was shown to specifically recognize chicken Hsp23 RNA by Northern (RNA) blot analysis and hybrid-select translation. This probe was then used to measure chicken hsp23 gene activity in control and heat-stressed cells. The hsp23 gene(s) was transcriptionally active in non-heat-stressed cells, and its rate of transcription did not increase significantly upon heat shock. Cytoplasmic Hsp23 mRNA, which was metabolically very stable in nonstressed cells, underwent a fourfold increase in amount after a 1-h heat shock, resulting in a twofold increase in Hsp23 mRNA in polysomes. Hsp23 mRNA was relatively abundant and translationally active even in non-heat-shocked cells. Taken together, these data implicated posttranscriptional nuclear events as an important control point for induction of Hsp23 RNA transcripts. The protein half-life of Hsp23 increased from approximately 2 h in control cultures to 13 h in heat-shocked cells, revealing a second major control point. Hsp23 which was synthesized prior to heat shock also increased in stability and contributed to the overall accumulation of Hsp23 in heat-shocked cells. Cycloheximide had no effect on this change in Hsp23 half-life, while dactinomycin blocked the stabilization of Hsp23, suggesting a need for newly synthesized RNA. These data indicated that stabilization of Hsp23 protein and posttranscriptional nuclear events resulting in increased production of Hsp23 mRNA were primarily responsible for a 13-fold increase in the accumulation of newly synthesized Hsp23 after 1 h of heat shock. The regulation of the hsp23 gene is discussed in comparison with several other posttranscriptionally regulated genes, including the proto-oncogene c-fos, the developmentally regulated chicken delta-crystallin gene, and regulation of cellular gene expression by the proto-oncogene c-myc.
Mol Cell Biol 1990 Sep
PMID:Induction of a chicken small heat shock (stress) protein: evidence of multilevel posttranscriptional regulation. 238 29

During progesterone-induced maturation of the Rana temporaria oocytes phosphorylation of RNA(heparin)-binding proteins changes drastically due to the alterations in the activity of RNA-binding casein kinase II. Its activity increases 7 hr later administration of progesterone and correlates with the level of translation in oocytes. Cycloheximide almost completely inhibits the protein biosynthesis but has no effect on the activity of RNA-binding casein kinase and the set of phosphorylated polypeptides. The possibility is discussed that this enzyme participates in the translation control mechanisms.
Mol Biol (Mosk)
PMID:[Progesterone-induced activation of RNA-binding casein kinase type II during maturation of frog oocytes]. 245 64

To investigate differences in growth hormone (GH) and estrogen activation of insulin-like growth factor-I (IGF-I) expression, we have examined the effects of cycloheximide on hepatic and uterine IGF-I mRNA abundance in response to GH and 17 beta-estradiol (E2) respectively. In hypophysectomized (hypox) rats a single injection of GH significantly increased hepatic IGF-I mRNA 3.65 +/- 0.68-fold, P less than 0.005, 6 h after injection. Administration of cycloheximide 30 min prior to GH injection completely abolished this response. In contrast, in pituitary intact rats killed 6 h after administration of cycloheximide, hepatic IGF-I mRNA abundance was not significantly different from untreated control rats although the serum IGF-I concentration was significantly reduced; 119.9 +/- 11.8 vs. 270.2 +/- 48.7 ng/ml, P less than 0.005. In immature rats, injection of E2 (1 micrograms/100 g body weight) significantly increased uterine IGF-I mRNA 4.1 +/- 0.4-fold. Cycloheximide did not block the E2-induced increase in IGF-I mRNA but rather significantly enhanced the IGF-I response. These data indicate that continuing protein synthesis is required for GH induction of hepatic IGF-I mRNA in the hypox rat but is not required for E2 induction of uterine IGF-I mRNA. Furthermore, in pituitary-intact rats protein synthesis is not required for maintenance of hepatic IGF-I mRNA levels.
Mol Cell Endocrinol 1989 Jun
PMID:Effects of cycloheximide on hepatic and uterine insulin-like growth factor-I mRNA. 247 68

We have examined the role of rapidly turning over proteins in the T3 regulation of multiple rat hepatic genes. T3 induction of the rapidly responsive mRNA-S14 was markedly inhibited by cycloheximide (1 mg/100 g BW) or emetine (3 mg/100 g) injected ip 30 min before T3 (mRNA-S14 concentration was only 35% of that in T3-treated controls 8.5 h after administration of either protein synthesis inhibitor, P less than 0.01). Cycloheximide exhibited a similar effect on each of five other more slowly responsive T3 regulated genes. When cycloheximide was given 10 h after T3, the expected T3-induced rise of mRNA-S7 activity was completely prevented, and for mRNA-S4 activity the anticipated rise was blunted to 40% of T3-treated control (P less than 0.05). Cycloheximide caused sharp declines in the activity of two other mRNAs, S6 and S8, which because of shorter lag times of response to T3, had already risen when the drug was given. Values for both these mRNAs returned to the baseline hypothyroid level within 6 h of injection of the drug and remained low for a further 8 h (P less than 0.05). The expected deinduction of mRNA-S10 by T3 was also markedly modified. T3 lowered this mRNA to 11% of the hypothyroid control after 8 h, whereas cycloheximide given 30 min before the hormone blunted this fall to only 72% of control (P less than 0.01). Thus there appeared to be a 70% reduction in the rate of T3 induced fall of mRNA-S10. We did not find that cycloheximide caused a generalized decrease in poly (A)+ RNA mass.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Endocrinol 1987 Jun
PMID:Triiodothyronine regulation of multiple rat hepatic genes: requirement for ongoing protein synthesis. 248 15


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