A proteomics approach for identifying osmotic-stress-related proteins in rice

Osmotic stress can endanger the survival of plants. To investigate the mechanisms of how plants respond to osmotic stress, rice protein profiles from mannitol-treated plants, were monitored using a proteomics approach. Two-week-old rice seedlings were treated with 400mM mannitol for 48h. After separation of proteins from the basal part of leaf sheaths by two-dimensional polyacrylamide gel electrophoresis, 327 proteins were detected. The levels of 12 proteins increased and the levels of three proteins decreased with increasing concentration or duration, of mannitol treatment. Levels of a heat shock protein and a dnaK-type molecular chaperone were reduced under osmotic, cold, salt and drought stresses, and ABA treatment, whereas a 26S proteasome regulatory subunit was found to be responsive only to osmotic stress. Furthermore, proteins whose accumulation was sensitive to osmotic stress are present in an osmotic-tolerant cultivar. These results indicate that specific proteins expressed in the basal part of rice leaf sheaths show a coordinated response to cope with osmotic stress.     

Differential Modulation of D1 and D2 Dopamine-Sensitive Adenylate Cyclases by 17β-Estradiol in Cultured Striatal Neurons and Anterior Pituitary Cells

Primary cultures of anterior pituitary cells from female rats and of mouse embryonic striatal neurons were used to study the effects of 17 beta-estradiol on D1- and D2-dopamine (DA)-sensitive adenylate cyclase. 17 beta-Estradiol pretreatment (10(-9) M, 72 h) suppressed the D2-DA-induced inhibition of adenylate cyclase activity in anterior pituitary cells. The steroid (10(-9) M, 24 h) also blocked the D2-DA-evoked response in striatal neurons whereas it enhanced by twofold the D1-DA-induced stimulation of the enzyme activity in these neurons. All these effects of the steroid were dose dependent and specific, as neither 17 alpha-estradiol, dexamethasone, nor progesterone used at the same concentration (10(-9) M) was effective. Furthermore, the modulation of DA-sensitive adenylate cyclases by the steroid required long-term exposure of living cells to 17 beta-estradiol since neither 17 beta-estradiol pretreatment for 4 h nor its addition to broken cells directly into the adenylate cyclase assay induced any alteration in the DA-sensitive adenylate cyclase activity. These results are in agreement with a genomic effect of the steroid. Using both anterior pituitary cells and striatal neurons in culture, 17 beta-estradiol affected neither the total number of DA (D1 and D2) receptors nor the estimated number of adenylate cyclase catalytic units. Therefore, it is suggested that the steroid modifies the coupling process by a mechanism that still has to be elucidated. These results demonstrate an effect of 17 beta-estradiol on DA target cells in both systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Impaired down-regulation of pituitary dopamine receptors by estradiol in aged rats

Administration of 17 beta-estradiol to mature (6-12 months) rats results in a more than 50% reduction in pituitary dopamine receptor concentrations, without affecting binding affinity. In contrast, when the same manipulation is performed on senescent (24-25 months) rats, negligible change in receptor concentration occurs. These results suggest that age-related increases in estrogen-stimulated prolactin release are not due to decreased dopaminergic inhibition at the receptor level.     

17beta-estradiol inhibits NADPH oxidase activity through the regulation of p47phox mRNA and protein expression in THP-1 cells

In this report, we demonstrate that NADPH oxidase is activated by tumor necrosis factor-alpha (TNF-alpha) plus interferon-gamma (IFN-gamma) in human monocytic cells (THP-1 cells) differentiated with phorbol ester (PMA) and that physiological concentration of 17beta-estradiol inhibits NADPH oxidase activity in THP-1 cells stimulated with TNF-alpha plus IFN-gamma. This effect is mediated by estrogen receptor based on estrogen receptor antagonist (ICI 182, 780) that diminishes inhibition by 17beta-estradiol. This inhibition is specific in 17beta-estradiol because 17alpha-estradiol, testosterone and progesterone do not inhibit NADPH oxidase activity. Activation of NADPH oxidase induced by TNF-alpha plus IFN-gamma is caused by up-regulation of p47(phox) (cytosolic component of NADPH oxidase) expression. 17beta-Estradiol prevents the up-regulation of p47(phox) mRNA and protein expression. This prevention of p47(phox) expression depends on the inhibition of NF-kappaB activation. Our results implicate that 17beta-estradiol has an anti-atherosclerotic effects through the improvement of nitric oxide (NO) bioavailability caused by the regulation of superoxide (O(2)(-)) production.     

Regulation of ovine pituitary glycoprotein hormone alpha subunit mRNA by 17 beta-estradiol in cell culture

Messenger RNA (mRNA) activity for the alpha glycoprotein subunit in ovine pituitary cell cultures was quantified using a wheat germ translation assay. [35S] Cysteine alpha translation product normally accounted for 0.3-0.9% of total 35S-product in translations of total culture RNA. Treatment of cultures with 1 nM 17 beta-estradiol (E2) decreased alpha mRNA activity by 50% and 92% after 1.8 and 8 days, respectively. Alpha mRNA activity decreased by 1/2 in 51 h, which is about three times slower than the rate of decrease reported for follicle-stimulating hormone (FSH) beta mRNA activity in similarly treated cultures (Alexander and Miller, 1982). The effective dose (ED50) for E2 in vitro was 30 pM (8 pg/ml), which is within the physiological range for ewes. These results indicate that E2 can act directly on the ovine pituitary to decrease alpha mRNA activity coordinately with mRNA activity for FSH beta.     

Estrogen stimulates both prolactin and growth hormone mRNAs expression in the MtT/F4 transplantable pituitary tumor

The effects of 17 beta-estradiol (E2) on MtT/F4 pituitary tumor growth and on prolactin (PRL) and growth hormone mRNA expression were analyzed in F344 female rats. E2 (10 mg) stimulated pituitary PRL cell hyperplasia and PRL mRNA, but inhibited growth of the transplantable tumors. The expression of both PRL and growth hormone mRNA levels was increased in the MtT/F4 tumors. The effects of E2 on increasing PRL mRNA levels were more marked in the pituitary compared with the tumors. These results indicate that estrogens stimulate proliferation and PRL expression in the pituitary while inhibiting cell proliferation in the MtT/F4 tumor. E2 also stimulated both growth hormone and PRL mRNA expression in the MtT/F4 transplantable tumor.     

Cordycepin and early effects of estradiol on the immature rat uterus.

Estradiol induces the synthesis of a specific protein fraction (IP) in the uterus of the immature rat. The injection of cordycepin (3' deoxyadenosine), an inhibitor of poly A synthesis, inhibits the synthesis of IP. This fact suggests that one of the earliest effects of estrogen is the production of Hn-RNA poly-A relative to IP. Moreover, using electron microscopy, the stimulation by estradiol of the nucleolus of the immature rat uterine epithelium has been shown. Cordycepin does not affect this stimulation to any appreciable extent. Biochemical studies (incorporation of radioactive stracers into NRA, affinity chromatography on poly U-Sepharose) carried out in parallel with and under conditions comparable to those used in electron microscopy show that cordycepin does not greatly affect the increase in ribosomal RNA observed under the effect of estradiol. The blocking of IP by cordycepin and the lack of inhibition at the nucleolus level under the same conditions, show that the two early effects of the action of estrogen on the immature rat uterus are not directly correlated.     

Long-term estradiol treatment improves VIP-mediated vasodilation in atherosclerotic proximal coronary arteries

The aim of the present study was to evaluate the impact of long-term estrogen replacement therapy (ERT) on the vasodilatory effect of the two peptides vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase activating polypeptide (PACAP) in atherosclerotic coronary and cerebral arteries.Female ovariectomized homozygous Watanabe heritable hyperlipidemic rabbits were randomized to 16 weeks treatment with 17beta-estradiol or placebo. The diet was semisynthetic, thereby avoiding the influence of phytoestrogens. Artery ring segments were mounted for isometric tension recordings in myographs. Following precontraction, the dose-response relationships for VIP and PACAP were evaluated.Treatment with 17beta-estradiol significantly improved the maximum VIP-mediated vasodilation (E(max), percentage of precontraction) in proximal coronary arteries (45.8+/-9.6% vs. 24.1+/-3.7%, p<0.05). In the same artery segment, 17beta-estradiol induced a significant decrease in the relative ratio between the repeated contractile response to potassium 30 and 120 mM (100+/-7% vs. 132+/-11%, p<0.05). For distal coronary arteries, there was a tendency to similar changes, but no statistical differences for the potassium or VIP responses in cerebral or distal coronary arteries were found between the two groups. 17beta-estradiol induced no changes in the PACAP-mediated vasodilation.These results suggest that long-term treatment with 17beta-estradiol improves the VIP-mediated but not the PACAP-mediated vasodilation in atherosclerotic proximal coronary arteries.     

17beta-estradiol attenuates programmed cell death in cortical pericontusional zone following traumatic brain injury via upregulation of ERalpha and inhibition of caspase-3 activation

Pericontusional zone (PCZ) of traumatic cerebral contusion is a target of pharmacological intervention. It is well studied that 17beta-estradiol has a protective role in ischemic brain injury, but its role in brain protection of traumatic brain damage deserves further investigation, especially in pericontusional zone. Here we show that 17beta-estradiol enhances the protein expression and mRNA induction of estrogen alpha receptor (ERalpha) and prevents from programmed cell death in cortical pericontusional zone. ERalpha specific antagonist blocks this protective effect of 17beta-estradiol. Caspase-3 activation occurs in cortical pericontusional zone of the oil-treated injured rat brain and its activation is inhibited by 17beta-estradiol treatment. Additionally, ERalpha specific antagonist reverses this inhibition. Pan-caspase inhibitor also protect cortical pericontusional zone from programmed cell death. Our present study indicates 17beta-estradiol protects from programmed cell death in cortical pericontusional zone via enhancement of ERalpha and decrease of caspase-3 activation.     

Insulin inhibition of apolipoprotein B mRNA translation is mediated via the PI-3 kinase/mTOR signaling cascade but does not involve internal ribosomal entry site (IRES) initiation

Although insulin normally activates global mRNA translation, it has a specific inhibitory effect on translation of apolipoprotein B (apoB) mRNA. This suggests that insulin induces a unique signaling cascade that leads to specific inhibition of apoB mRNA translation despite global translational stimulation. Recent studies have revealed that insulin functions to regulate apoB mRNA translation through a mechanism involving the apoB mRNA 5' untranslated region (5' UTR). Here, we further investigate the role of downstream insulin signaling molecules on apoB mRNA translation, and the mechanism of apoB mRNA translation itself. Transfection studies in HepG2 cells expressing deletion constructs of the apoB 5' UTR showed that the cis-acting region responding to insulin was localized within the first 64 nucleotides. Experiments using chimeric apoB UTR-luciferase constructs transfected into HepG2 cells followed by treatment with wortmannin, a PI-3K inhibitor, and rapamycin, an mTOR inhibitor, showed that signaling via PI-3K and mTOR pathways is necessary for insulin-mediated inhibition of chimeric 5' UTR-luciferase expression. In vitro translation of chimeric cRNA confirmed that the effects observed were translational in nature. Furthermore, using RNA-EMSA we found that wortmannin pretreatment blocked insulin-mediated inhibition of the binding of RNA-binding factor(s), migrating near the 110 kDa marker, to the 5' UTR. Radiolabeling studies in HepG2 cells also showed that insulin-mediated control of the synthesis of endogenously expressed full length apoB100 is mediated via the PI-3K and mTOR pathways. Finally, using dual-cistronic luciferase constructs we demonstrate that apoB 5' UTR may have weak internal ribosomal entry (IRES) translation which is not affected by insulin stimulation, and may function to stimulate basal levels of apoB mRNA translation.     

Effect of estradiol on the early incorporation of (3H) thymidine into uterine DNA on immature mouse.

The incorporation of [3H]thymidine into uterine DNA was markedly depressed within 10 to 30 minutes after intraperitoneal administration of 17beta-estradiol to immature mouse. Maximum inhibition occurred about 6 hours after the hormone was administered. Uterine DNA content and the amount of [3H]thymidine incorporated into the acid-soluble fraction was not affected during the period of hormone-induced inhibition. Moreover, the in vitro incorporation of [3H]thymidine by isolated estradiol-treated mouse uterus was blocked. In contrast to the uterus, 17beta-estradiol did not influence the incorporation of thymidine into mouse liver DNA. Evidence is presented to show that the incorporation of thymidine into uterine DNA was blocked initially by 17beta-estradiol.     

Abscisic-acid-induced turion formation in Spirodela polyrrhiza L III. Specific changes in protein synthesis and translatable RNA during turion development

Abstract The developmental process leading to the formation of the abscisic acid (ABA) induced turion of Spirodela polyrrhiza was accompanied by a repression of nucleic acid and protein synthesis. DNA synthesis in the developing lurion (induced by 10⁻⁴mol m⁻³ ABA) was inhibited within 3h of ABA addition, followed by a repression of protein synthesis after 24 h, while RNA synthesis was not inhibited until 3 d. The inhibitory effect of ABA on protein synthesis was found to be selective and the synthesis of several novel proteins appeared to be induced. These effects were specific to ABA-sensitive tissue. The relationship between the changes in the protein and mRNA profiles during the development of the turion was investigated. The rapid general inhibition of protein synthesis at early stages of lurion formation could not be accounted for by the level of translatable mRNA, indicating an effect of ABA at the translational level. The specific alteration to the pattern of in vivo labelled proteins could have resulted, however, from control of the level of specific mRNAs for those particular proteins. Only after 3 d in ABA, when the developing primordium is committed to the turion developmental pathway, is there a total inhibition in the production of mRNA leading to the shutdown of all primary processes and the onset of the irreversible events leading to the dormant state.     

In vitro study of the biological activity of RNAs after incubation of hog liver, heart and brain tissue at room temperature

The biological activity of RNA, isolated from tissue which was incubated for 1, 3, or 6 hours at room temperature (simulation of post-mortem conditions), was preserved. However, the different organs used differ from each other. When liver is used, qualitative differences in the in vitro translation products are observed, after one hour incubation at room temperature, whereas when heart and brain are used these differences are not observed. We have also shown that relatively small amounts of post-mortem tissue is sufficient for RNA extraction. When using frozen tissue it is absolutely necessary to add RNase inhibitors during thawing to reduce the loss of biological activity.     

Binding of 17-alpha-ethinyl estradiol and mestranol to uterine and pituitary gland receptors of the rat in vitro

Comparative study, through radiochemical analysis, of the binding of estrogens to the uterine and pituitary receptors in immature rats showed that, for binding to the uterine recpetors, a free OH group in Position 3 is necessary. 17alpha-ethinyl estradiol as well as mestranol (17alpha-ethinyl estradiol-3-methyl ether), showed a stronger affinity for the pituitary than the uterine receptors. Indeed, mestranol, among the 3-methyl ether estrogens, has a special place in respect to its affinity for the pituitary. Since the binding to the receptors is an essential stage in inhibition or stimulation effects, this is a significant point. It may be that mestranol exercises its recognized central effect not only indirectly through release of ethinyl estradiol, but also directly.     

Localized Rho GTPase activation regulates RNA dynamics and compartmentalization in tumor cell protrusions

mRNA trafficking and local protein translation are associated with protrusive cellular domains, such as neuronal growth cones, and deregulated control of protein translation is associated with tumor malignancy. We show here that activated RhoA, but not Rac1, is enriched in pseudopodia of MSV-MDCK-INV tumor cells and that Rho, Rho kinase (ROCK), and myosin II regulate the microtubule-independent targeting of RNA to these tumor cell domains. ROCK inhibition does not affect pseudopodial actin turnover but significantly reduces the dynamics of pseudopodial RNA turnover. Gene array analysis shows that 7.3% of the total genes analyzed exhibited a greater than 1.6-fold difference between the pseudopod and cell body fractions. Of these, only 13.2% (261 genes) are enriched in pseudopodia, suggesting that only a limited number of total cellular mRNAs are enriched in tumor cell protrusions. Comparison of the tumor pseudopod mRNA cohort and a cohort of mRNAs enriched in neuronal processes identified tumor pseudopod-specific signaling networks that were defined by expression of M-Ras and the Shp2 protein phosphatase. Pseudopod expression of M-Ras and Shp2 mRNA were diminished by ROCK inhibition linking pseudopodial Rho/ROCK activation to the localized expression of specific mRNAs. Pseudopodial enrichment for mRNAs involved in protein translation and signaling suggests that local mRNA translation regulates pseudopodial expression of less stable signaling molecules as well as the cellular machinery to translate these mRNAs. Pseudopodial Rho/ROCK activation may impact on tumor cell migration and metastasis by stimulating the pseudopodial translocation of mRNAs and thereby regulating the expression of local signaling cascades.     

Evidence for the synthesis of multiple pro-opiomelanocortin-like precursors in murine Leydig tumor cells

The opiate peptide beta-endorphin has recently been localized in extrapituitary tissues and cells, including the Leydig cells of the testis, by immunohistochemical techniques. An intriguing question is whether this localization reflects an accumulation of the peptide through a specific uptake mechanism or is the result of synthesis within the cell in a large precursor form similar to pro-opiomelanocortin synthesized in the pituitary. Evidence is presented herein that specific antibodies against beta-endorphin and adrenocorticotrophic hormone precipitate identical precursor molecules from total cellular mRNA translation products of M5480A Leydig tumor cells. In addition, mRNA from these cells cross-hybridizes under stringent conditions with a cDNA coding for the rat pituitary pro-opiomelanocortin sequence. These data demonstrate the synthesis of a pro-opiomelanocortin-like mRNA in this Leydig cell tumor and strongly implicate biosynthesis within these cells.