Changes In Steady-state Levels Research Articles

MRNA stability changes precede changes in steady-state mRNA amounts during hyperosmotic stress

Under stress, cells need to optimize the activity of a wide range of gene products during the response phases: shock, adaptation, and recovery. This requires coordination of several levels of regulation, including turnover and translation efficiencies of mRNAs. Mitogen-activated protein (MAP) kinase pathways are implicated in many aspects of the environmental stress response, including initiation of transcription, translation efficiency, and mRNA turnover. In this study, we analyze mRNA turnover rates and mRNA steady-state levels at different time points following mild hyperosmotic shock in Saccharomyces cerevisiae cells. The regulation of mRNA stability is transient and affects most genes for which there is a change in transcript level. These changes precede and prepare for the changes in steady-state levels, both regarding the initial increase and the later decline of stress-induced mRNAs. The inverse is true for stress-repressed genes, which become stabilized during hyperosmotic stress in preparation of an increase as the cells recover. The MAP kinase Hog1 affects both steady-state levels and stability of stress-responsive transcripts, whereas the Hog1-activated kinase Rck2 influences steady-state levels without a major effect on stability. Regulation of mRNA stability is a wide-spread, but not universal, effect on stress-responsive transcripts during transient hyperosmotic stress. By destabilizing stress-induced mRNAs when their steady-state levels have reached a maximum, the cell prepares for the subsequent recovery phase when these transcripts are to return to normal levels. Conversely, stabilization of stress-repressed mRNAs permits their rapid accumulation in the recovery phase. Our results show that mRNA turnover is coordinated with transcriptional induction.

The interaction of beta-amyloid protein with cellular membranes stimulates its own production

Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ (1–40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ (1–40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.

Regulation of Sertoli Cell Steroidogenic Acute Regulatory (StAR)-Related Lipid Transfer (START-Domain-Containing) StARD1 and StARD4 Expression by Follicle Stimulating Hormone (FSH) Requires Multiple Protein Kinases.

Multiple mechanisms mediate gonadotropin (follicle stimulating hormone: FSH) regulation of Sertoli cell (SC) gene expression, although the temporal coordination of signalling components other than cAMP remain largely unknown. Cytokines within the seminiferous tubule participate in such physiological regulation, subsequently influencing germ cell development. Recently, we demonstrated differential regulation of Sertoli START-domain-containing proteins steroidogenic acute regulatory (StAR)-related lipid transfer (START-domain-containing) StARD1 and D5, but not D4, in response to cJun-N-terminal kinase (JNK) activation by interleukin (IL)-1beta. Herein differential START gene regulatory mechanisms in this specialized epithelial cell were further examined. In a time- and dose-dependent manner, both FSH and forskolin significantly increase expression of StARD1 and D4 mRNAs, but not that of D5. Whereas StARD1 protein increased in response to FSH and forskolin, no changes in steady state levels of D4 or D5 were observed. Unlike IL-1beta, FSH or forskolin increases in cAMP rapidly results in phosphorylation of cAMP response element binding protein, protein kinase C (PKC) and phosphatidylinositol 3 kinase (PI3K), but not JNK or p38 mitogen-activated protein kinase (MAPK). Also, in contrast to IL-1beta, neither FSH nor forskolin induces cyclooxygenase activity or prostaglandin (PG)E2 or PGF2alpha production. Interestingly, FSH and forskolin reduce basal phosphorylated extracellular signal regulated kinase levels. When PKA, PKC, or PI3K inhibitors are used with FSH or forskolin, induction of StARD1 and D4 expression is inhibited, consistent with transcriptional mechanisms dependent, in part, on activation of several kinases. The data demonstrate that Sertoli StARD1, D4 and D5 expression are differentially regulated. Testicular START-domain proteins may provide novel contraceptive targets or therapeutic interventions in male infertility secondary to lipid handling pathologies.

Manganese superoxide dismutase gene dosage affects chromosomal instability and tumor onset in a mouse model of T cell lymphoma

Increased reactive oxygen species (ROS) such as superoxide have been implicated as causal elements of oncogenesis. A variety of cancers have displayed changes in steady-state levels of key antioxidant enzymes, with the mitochondrial form of superoxide dismutase (MnSOD) being commonly implicated. Increasing MnSOD expression suppresses the malignant phenotype in various cancer cell lines and suppresses tumor formation in xenograft and transgenic mouse models. We examined the impact of MnSOD expression in the development of T cell lymphoma in mice expressing proapoptotic Bax. Lck-Bax38/1 transgenic mice were crossed to mice overexpressing MnSOD (Lck-MnSOD) as well as MnSOD+/− mice. The effects of MnSOD on apoptosis, cell cycle, chromosomal instability (CIN), and lymphoma development were determined. The apoptotic and cell cycle phenotypes observed in thymocytes from control and Bax transgenic mice were unaffected by variations in MnSOD levels. Remarkably, increased gene dosage of MnSOD significantly decreased aneuploidy in premalignant thymocytes as well as the onset of tumor formation in Lck-Bax38/1 mice. The observed effects of MnSOD support a role for ROS in CIN and tumor formation in this mouse model of T cell lymphoma.

Abstract for the Forthcoming International Federation of Placenta Associations Meetings (13th Meeting) in Kingston, Ontario, Canada

Neuroblastoma is the most common extra-cranial solid tumor in childhood; and patients in stage IV of the disease have a high propensity for tumor recurrence. Retinoid therapy has been utilized as a means to induce differentiation of tumor cells and to inhibit relapse. In this study, the expression of a common neuronal differentiation marker [neurofilament M (NF-M)] in human SK-N-SH neuroblastoma cells treated with 10 μM all-trans retinoic acid (ATRA) showed significantly increased expression in accordance with reduced cell number. This was accompanied by an increase in MitoSOX and DCFH2 oxidation that could be indicative of increased steady-state levels of reactive oxygen species (ROS) such as O2•− and H2O2, which correlated with increased levels of MnSOD activity and immuno-reactive protein. Furthermore PEG-catalase inhibited the DCFH2 oxidation signal to a greater extent in the ATRA-treated cells (relative to controls) at 96 h indicating that as the cells became more differentiated, steady-state levels of H2O2 increased in the absence of increases in peroxide-scavenging antioxidants (i.e., glutathione, glutathione peroxidase, and catalase). In addition, ATRA-induced stimulation of NF-M at 48 and 72 h was enhanced by decreasing SOD activity using siRNA directed at MnSOD. Finally, treatment with ATRA for 96 h in the presence of MnSOD siRNA or PEG-catalase inhibited ATRA induced increases in NF-M expression. These results provide strong support for the hypothesis that changes in steady-state levels of O2•− and H2O2 significantly contribute to the process of ATRA-induced differentiation in neuroblastoma, and suggest that retinoid therapy for neuroblastoma could potentially be enhanced by redox-based manipulations of superoxide metabolism to improve patient outcome.

Oxidative stress in the aging murine olfactory bulb: Redox proteomics and cellular localization

A recent proteomics analysis from our laboratory demonstrated that several oxidative stress response proteins showed significant changes in steady-state levels in olfactory bulbs (OBs) of 20- vs. 1.5-month-old mice. Oxidative stress may result in protein oxidation. In this study, we investigated two forms of protein oxidative modification in murine OBs: carbonylation and nitration. Redox proteomics with two-dimensional gel electrophoresis, Western blotting, protein digestion, and mass spectrometry was used to quantify total and specific protein carbonylation and to identify differentially carbonylated proteins and determine the carbonylation status of previously identified proteins in OBs of 1.5- and 20-month-old mice. Immunohistochemistry was used to demonstrate the relative intensity and localization of protein nitration in OBs of 1.5-, 6-, and 20-month-old mice. Total protein carbonylation was significantly greater in OBs of 20- vs. 1.5-month-old mice. Aldolase 1 (ALDO1) showed significantly more carbonylation in OBs from 20- vs. 1.5-month-old mice; heat shock protein 9A and dihydropyrimidinase-like 2 showed significantly less. Several previously investigated proteins were also carbonylated, including ferritin heavy chain (FTH). Nitration, identified by 3-nitrotyrosine immunoreactivity, was least abundant at 1.5 months, intermediate at 6 months, and greatest at 20 months and was localized primarily in blood vessels. Proteins that were specific targets of oxidation were also localized: ALDO1 in astrocytes of the granule cell layer and FTH in mitral/tufted cells. These results indicate that specific carbonylated proteins, including those in astrocytes and mitral/tufted neurons, and nitrated proteins in the vasculature are molecular substrates of age-related olfactory dysfunction.

Human Chorionic Gonadotropin-Dependent Up-Regulation of Genes Responsible for Estrogen Sulfoconjugation and Export in Granulosa Cells of Luteinizing Preovulatory Follicles

Estrogen sulfotransferase (EST) is responsible for the sulfoconjugation of estrogens, thereby changing their physical properties and preventing their action via the estrogen receptors. These sulfoconjugated steroids no longer diffuse freely across the lipid bilayer; instead, they are exported by members of the ATP-binding cassette family, such as ABCC1. The objective of this study was to investigate the regulation of EST and ABCC1 during human chorionic gonadotropin (hCG)-induced ovulation/luteinization. The transcripts for EST and ABCC1 were cloned by RT-PCR, and the regulation of their mRNAs was studied in preovulatory follicles obtained during estrus at 0, 12, 24, 30, 33, 36, and 39 h after hCG. Results obtained from RT-PCR/Southern blot analyses showed significant changes in steady-state levels of both EST and ABCC1 mRNA after hCG treatment (P < 0.05). In granulosa cells, a significant increase in EST transcript was observed 30-39 h after hCG. Similarly, ABCC1 transcript levels were induced in granulosa cells 12-39 h after hCG. In contrast, no significant changes in either EST or ABCC1 were detected in theca interna samples after hCG. The increase in EST and ABCC1 transcripts observed in granulosa cells was reflected in preparations of intact follicle walls, suggesting that the granulosa cell layer contributes the majority of EST and ABCC1 expression in preovulatory follicles. The present study demonstrates that follicular luteinization is accompanied not only by a decrease in 17 beta-estradiol biosynthesis but also by an increase in expression of genes responsible for estrogen inactivation and elimination from granulosa cells, such as EST and ABCC1, respectively.

M1 Receptors Play a Central Role in Modulating AD-like Pathology in Transgenic Mice

We investigated the therapeutic efficacy of the selective M1 muscarinic agonist AF267B in the 3xTg-AD model of Alzheimer disease. AF267B administration rescued the cognitive deficits in a spatial task but not contextual fear conditioning. The effect of AF267B on cognition predicted the neuropathological outcome, as both the Abeta and tau pathologies were reduced in the hippocampus and cortex, but not in the amygdala. The mechanism underlying the effect on the Abeta pathology was caused by the selective activation of ADAM17, thereby shifting APP processing toward the nonamyloidogenic pathway, whereas the reduction in tau pathology is mediated by decreased GSK3beta activity. We further demonstrate that administration of dicyclomine, an M1 antagonist, exacerbates the Abeta and tau pathologies. In conclusion, AF267B represents a peripherally administered low molecular weight compound to attenuate the major hallmarks of AD and to reverse deficits in cognition. Therefore, selective M1 agonists may be efficacious for the treatment of AD.

Oxidative phosphorylation dysfunction modulates expression of extracellular matrix—remodeling genes and invasion

A number of recent studies suggest that mitochondrial function is a player in tumor development and progression. In this study, we have used gene expression arrays to examine transcriptional differences between oxidative phosphorylation (OXPHOS)-competent and OXPHOS-impaired human osteosarcoma cells. Genes associated with extracellular matrix remodeling, including members of the matrix metalloproteinases (MMPs) and tissue inhibitors of the MMP (TIMP) family, urokinase plasminogen activator and its inhibitor plasminogen-activator inhibitor-1 (PAI1), and CTGF and CYR61 (members of the Cysteine-rich 61, Connective Tissue Growth Factor and Nephroblastoma-overexpressed (CCN) gene family of growth regulators), were among the ones significantly altered in the OXPHOS-deficient cells. These changes were confirmed by RT-PCR and promoter reporter assays. Alterations at the protein level for some of these factors were also observed, though at a lower magnitude, with the exception of TIMP1, where a marked change in steady-state levels of the protein was observed after induction of OXPHOS dysfunction. Repopulation of mitochondrial DNA (mtDNA)-less cells with wild-type mtDNA reduced matrigel invasion, whereas repopulation with a mutated mtDNA did not. Taken together our data suggests that OXPHOS dysfunction modulates the invasive phenotype by transcriptional regulation of genes coding for members of the MMP/TIMP system, urokinase plasminogen activator/plasminogen-activator inhibitor I and CCN proteins.

Changes in steady-state concentrations of messenger ribonucleic acids in luteal tissue during prostaglandin F2α induced luteolysis in mares

Transvaginal ultrasound-guided luteal biopsy was used to evaluate the effects of prostaglandin (PG)F2α on steady-state concentrations of mRNA for specific genes that may be involved in regression of the corpus luteum (CL). Eight days after ovulation (Hour 0), mares ( n = 8/group) were randomized into three groups: control (no treatment or biopsy), saline + biopsy (saline treatment at Hour 0 and luteal biopsy at Hour 12), or PGF2α + biopsy (5 mg PGF2α at Hour 0 and luteal biopsy at Hour 12). The effects of biopsy on CL were compared between the controls (no biopsy) and saline + biopsy group. At Hour 24 (12 h after biopsy) there was a decrease in circulating progesterone in saline group to 56% of pre-biopsy values, indicating an effect of biopsy on luteal function. Mean plasma progesterone concentrations were lower ( P < 0.001) at Hour 12 in the PG group compared to the other two groups. The relative concentrations of mRNA for different genes in luteal tissue at Hour 12 was quantified by real time PCR. Compared to saline-treated mares, treatment with PGF2α increased mRNA for cyclooxygenase-2 (Cox-2, 310%, P < 0.006), but decreased mRNA for LH receptor to 44% ( P < 0.05), steroidogenic acute regulatory protein to 22% ( P < 0.001), and aromatase to 43% ( P < 0.1) of controls. There was no difference in mRNA levels for PGF2α receptor between PG and saline-treated groups. Results indicated that luteal biopsy alters subsequent luteal function. However, the biopsy approach was effective for collecting CL tissue for demonstrating dynamic changes in steady-state levels of mRNAs during PGF2α-induced luteolysis. Increased Cox-2 mRNA concentrations suggested that exogenous PGF2α induced the synthesis of intraluteal PGF2α. Thus, the findings are consistent with the concept that an intraluteal autocrine loop augments the luteolytic effect of uterine PGF2α in mares.

Molecular characterization and expression studies during melon fruit development and ripening of L-galactono-1,4-lactone dehydrogenase.

The last step of ascorbic acid (AA) biosynthesis is catalysed by the enzyme L-galactono-1,4-lactone dehydrogenase (GalLDH, EC 1.3.2.3), located on the inner mitochondrial membrane. The enzyme converts L-galactono-1,4-lactone to ascorbic acid (AA). In this work, the cloning and characterization of a GalLDH full-length cDNA from melon (Cucumis melo L.) are described. Melon genomic DNA Southern analysis indicated that CmGalLDH was encoded by a single gene. CmGalLDH mRNA accumulation was detected in all tissues studied, but differentially expressed during fruit development and seed germination. It is hypothesized that induction of CmGalLDH gene expression in ripening melon fruit contributes to parallel increases in the AA content and so playing a role in the oxidative ripening process. Higher CmGalLDH message abundance in light-grown seedlings compared with those raised in the dark suggests that CmGalLDH expression is regulated by light. Finally, various stresses and growth regulators resulted in no significant change in steady state levels of CmGalLDH mRNA in 20-d-old melon seedlings. To the authors' knowledge, this is the first report of GalLDH transcript induction in seed germination and differential gene expression during fruit ripening.

Primary sequence and secondary structure motifs in spleen necrosis virus RU5 confer translational utilization of unspliced human immunodeficiency virus type 1 reporter RNA.

The 5' long terminal repeat (LTR) of spleen necrosis virus (SNV) contains a unique posttranscriptional control element that facilitates Rev/Rev-responsive element-independent expression of unspliced human immunodeficiency virus type 1 (HIV-1) gag reporter RNA. HIV-1 Gag expression is eliminated when SNV LTR is repositioned to the 3' untranslated region or when the RU5 region is positioned in the antisense orientation. RU5 corresponds to the 5' RNA terminus, and results presented here indicate that Gag production is sustained upon introduction of transcribed spacers that reposition SNV RU5 35 to 200 nucleotides downstream. Concordant results of deletion and point mutagenesis identified two functionally redundant and synergistic motifs (designated A and C) that are necessary and sufficient for SNV RU5 activity. Enzymatic analysis of SNV RU5 RNA structure determined that A and C correspond to stem-loop structures. Quantitative RNA and protein analysis of A and C mutants revealed that the structural integrity of A and C is necessary for protein production, and loss of function correlates with little change in steady-state level, splicing efficiency, or cytoplasmic accumulation of HIV-1 gag reporter RNA. Instead, the structural mutations eliminate cytoplasmic utilization as an mRNA template for Gag protein production. Point mutations of unpaired loop-and-bulge nucleotides that maintain the structure of A eliminate activity. The results show that the unpaired UUGU loop and U-rich bulges function together and are candidate SNV RU5 binding sites for the host cell protein(s) that directs cytoplasmic utilization of unspliced HIV-1 reporter RNA.

Expression profiles of novel thyroid hormone-responsive genes and proteins in the tail of Xenopus laevis tadpoles undergoing precocious metamorphosis.

Thyroid hormones (THs) are critical for the growth, development, and homeostasis of many organisms and are necessary for metamorphosis of Xenopus laevis tadpoles. TH-induced metamorphosis requires alterations in the transcriptome and the proteome. However, only a few of the molecular components of this developmental program have been identified and their interrelationship remains unclear. Using a cDNA array comprised of 420 known anuran genes and quantitative PCR, we have identified 93 TH-responsive genes in the tail of premetamorphic tadpoles after exogenous administration of T3. Fifty-three of these mRNA transcripts have not previously been characterized as TH responsive in any species. The gene expression profiles show distinctive temporal patterns with most transcript steady-state levels increasing after induction of metamorphosis. Two-dimensional gel electrophoresis of total protein extracts from the tail shows changes in steady-state levels of many proteins after T3 treatment. Of the up-regulated proteins, 10 were identified by peptide mass mapping. These data identify potential components involved in the regulation of Xenopus tail regression by T3 and begin to address a critical question regarding the interrelationship between the transcriptome and the proteome in TH-dependent developmental processes.

The use of synthetic oligonucleotides as protein inhibitors and anticode drugs in cancer therapy: accomplishments and limitations.

The function of gene products can be altered at many levels, including the mutation of gene sequence and the change in steady state levels of mRNA and/or protein by various mechanisms. The cumulative malfunction of specific gene products underlies many pathological conditions such as the multi-step and multi-cause acquisition of cancer. Here we discuss two oligonucleotide-based strategies in which these compounds target defective gene products acting either as antiprotein or anticode agents. The SELEX technique (systematic evolution of ligands by exponential enrichment) is an antiprotein approach in which nuclease-resistant DNA or RNA aptamers are selected by their ability to bind their protein targets with high affinity and specificity of the same range as antibodies. Such inhibitors were previously evolved against a great variety of targets, including receptors, growth factors and adhesion molecules implicated in the genesis of some kinds of cancer. Moreover, some results have already been obtained in animal models. The antigene technology interferes with earlier steps in the information flow leading from gene to protein. In this approach selective gene silencing is provided by the formation of stable and specific complexes between triplex forming molecules and their DNA targets. The feasibility of this strategy as well as a molecular mechanism for the action of antigene oligonucleotides has been demonstrated in cellular systems and in vivo. The use of oligonucleotide drugs (of either the antiprotein or the anticode type) as a viable approach to cancer therapy is limited by some common problems that will be discussed.

Cyclic AMP in Ovarian Cancer Cells Both Inhibits Proliferation and Increases c-KIT Expression

C-KIT encodes a tyrosine kinase receptor (KIT) that, when activated by its ligand (KL), stimulates proliferation, differentiation, migration, and survival. Greater than 70% of epithelial ovarian cancers coexpress c-KIT and KL. C-KIT and KL expression levels have been shown to be up-regulated by cAMP in some cell types. Additionally, cAMP is well-recognized for its anti-proliferative effects in cancer cells. The goal of these experiments was to investigate these seemingly contradictory consequences of cAMP treatment by: (1) confirming the growth inhibitory actions of cAMP on ovarian cancer cells; (2) investigating the ability of cAMP to affect c-KIT and KL expression in these cells; and (3) examining the possible role of endogenous and/or cAMP-regulated c-KIT and KL expression in ovarian cancer cell proliferation. HEY cells, an ovarian cancer cell line which expresses c-KIT and KL, were treated with dibutyryl cyclic AMP (dbcAMP), 8-bromo-cAMP, and cholera toxin over a range of concentrations. With all treatments, stimulation of cAMP signaling caused a dose-dependent inhibition of HEY cell proliferation by up to 40, 62, and 38%, respectively. This inhibition of proliferation correlated with a dose-dependent increase in c-KIT mRNA expression, yielding 4- to 7-fold elevations in transcript abundance; there were no changes in steady-state levels of KL transcripts. In order to determine whether KIT expression/activity was responsible for the observed decrease in proliferation, dbcAMP-treated HEY cells were exposed either to anti-KIT neutralizing antibodies or to the KIT inhibitor STI571. These experiments demonstrated that KIT inhibition did not alter the growth rate of cells or reverse the dbcAMP-induced inhibition of proliferation. These results suggest that cAMP signaling pathways regulate both cell proliferation and c-KIT expression in ovarian cancer cells; however, KIT is not assuming its well-established role as a growth factor.

Auxin modulates the degradation rate of Aux/IAA proteins.

Aux/IAA gene family members were first identified by their rapid transcriptional increase in response to auxin. Auxin/indole-3-acetic acid protein (Aux/IAA) luciferase (LUC) fusions expressed in Arabidopsis under control of a non-auxin-responsive promoter were used to monitor the effect of auxin on protein abundance independent of transcriptional regulation by auxin. After 2 hr in the presence of 1 microM exogenous dichlorophenoxyacetic acid (2,4D), a synthetic auxin, the levels of pea IAA6 (PSIAA6) and Arabidopsis IAA1 LUC activity were 35% and 67%, respectively, of mock-treated genetically identical seedlings, whereas the activity of LUC alone from equivalently treated seedlings remained unaltered. The steady-state level of an Aux/IAA fusion protein lacking domain II, one of the conserved domains found in all Aux/IAA proteins, was not reduced in the presence of auxin. Higher levels of exogenous auxin were required to affect the steady-state level of the PSIAA6LUC fusion with a point mutation in domain II. A 13-aa consensus sequence from domain II fused to LUC created an auxin-responsive fusion protein. The change in steady-state levels in response to auxin is extremely rapid, with a decrease in LUC activity detectable by 2 min after auxin application. Direct half-life measurements show that the decrease caused by exogenous auxin is due to the decrease in fusion protein half-life. These results suggest that auxin rapidly modulates the degradation rate of Aux/IAA proteins, with higher levels of auxin increasing the proteolytic rate of Aux/IAA family members.

Neuroendocrine factors affecting the steady-state levels of early trypsin mRNA in Aedes aegypti

Transcription of the early trypsin gene occurs in the midgut after adult emergence under control of juvenile hormone (JH). We tested the hypothesis that factors that affect the steady-state levels of early trypsin mRNA do so by influencing the levels of JH. We investigated the effect of ingesting different meals on early trypsin mRNA levels as well as on JH levels. We also studied how early trypsin mRNA levels changed when the midgut was isolated from different components of the neuroendocrine system by abdominal ligation and decapitation. Early trypsin transcripts levels are high in unfed females; feeding different meals had three distinct effects on the changes of steady-state levels of early trypsin mRNA: (1) blood and protein meals caused the level to decrease drastically and remained low for at least 24 h; (2) amino acid meals caused a transient decrease in the mRNA level, but it returned to high levels after 12–18 h; and (3) sugar, latex and saline meals had no effect on the early trypsin mRNA steady-state levels. The changes in JH levels after ingesting blood and amino acid meals show profiles resembling the changes in early trypsin mRNA levels for the corresponding meal. Decapitation at 1, 2 and 3 days after emergence does not affect the steady-state levels of early trypsin in unfed females. In contrast, 24 h after feeding, transcript levels were significantly higher in decapitated females when compared with non-decapitated fed females. We propose that the changes in the steady-state levels of early trypsin mRNA observed after the ingestion of different meals, ligations and decapitations are generated by changes in the levels of juvenile hormone.

Dopamine-Deficient Mice Are Hypersensitive to Dopamine Receptor Agonists

Dopamine-deficient (DA-/-) mice were created by targeted inactivation of the tyrosine hydroxylase gene in dopaminergic neurons. The locomotor activity response of these mutants to dopamine D1 or D2 receptor agonists and l-3,4-dihydroxyphenylalanine (l-DOPA) was 3- to 13-fold greater than the response elicited from wild-type mice. The enhanced sensitivity of DA-/- mice to agonists was independent of changes in steady-state levels of dopamine receptors and the presynaptic dopamine transporter as measured by ligand binding. The acute behavioral response of DA-/- mice to a dopamine D1 receptor agonist was correlated with c-fos induction in the striatum, a brain nucleus that receives dense dopaminergic input. Chronic replacement of dopamine to DA-/- mice by repeated l-DOPA administration over 4 d relieved the hypersensitivity of DA-/- mutants in terms of induction of both locomotion and striatal c-fos expression. The results suggest that the chronic presence of dopaminergic neurotransmission is required to dampen the intracellular signaling response of striatal neurons.

9-cis-Retinoic Acid Decreases the Level of Its Cognate Receptor, Retinoid X Receptor, through Acceleration of the Turnover

In this study, we investigated the ligand-mediated regulation of retinoid X receptor (RXR) in two human cell lines (HepG2 and JEG-3 cells), which have been reported to express RXRα predominantly. Western blot analysis revealed that a treatment with 1 μM 9-cis-retinoic acid (9-cis RA) for 24 h decreased RXRα protein level to 72 ± 9 and 75 ± 7% in HepG2 and JEG-3 cells, respectively, when the levels in the non-treated cells were expressed as 100%. The decrease was not due to the changes in steady-state level of RXRα mRNA or its stability as revealed by Northern blot analysis. However, the 9-cis RA treatment decreased the half-life of RXRα protein as determined by pulse-chase analysis. It was thus demonstrated that 9-cis RA downregulates RXRα by increasing the turnover of the protein in the two cell lines. The ligand-dependent downregulation of RXRα protein may be important for several hormonal signalings, in which the receptors heterodimerize with RXR.

Effects of Nitric Oxide Synthase Blockade on the Acute Response of the Reproductive Axis to Ethanol in Pubertal Male Rats

The effects of ethanol (EtOH) and nitric oxide (NO) are well known in the adult male rat reproductive axis. In the present study, we investigate the effects of EtOH, NO, and their interaction on key genes and reproductive hormone levels in mid-(45-day) and late pubertal(55-day) male rats. Using three different NO synthase blockers—N-nitro-L-arginine methyl ester (L-NAME), N;-nitro-L-arginine (L-NA), and 7-nitroi-ndazole—we show that it is possible to block, in part, some of the disruptive effects of EtOH. L-NAME totally prevented the EtOH-induced fall in serum testosterone in both 45- and 55-day-old rats (p < 0.05 and p < 0.001, respectively). On the other hand, the D-NAME, an inactive isomer of L-NAME, did not protect testosterone from suppression caused by EtOH. Similarly, L-NA and 7-nitroindazole prevented the suppression of testosterone caused by EtOH in 55-day-old animals (p < 0.001 L-NA and p <0.05 for 7-nitroindazole), but not in the 45-day-old rats. Serum luteinizing hormone (LH) was significantly reduced by EtOH in all the studies in both age groups. L.-NAME (but not D-NAME) and L-NA prevented this inhibition in 55-day-old animals (p < 0.001 for L-NAME and p < 0.01 for L-NA). However, only L-NA was able to prevent the effects of EtOH on LH in the 45-day-old rats. 7-Nitroindazole was unable to prevent the decrease in LH in either age group. Despite changes in the other reproductive hormones, there were no consistent changes in hypothalamic concentrations of either LH releasing hormone (LHRH) or its precursor. pro-LHRH. No treatment caused any change in steady-state levels of β-LH mRNA. There were no consistent changes in pro-LHRH mRNA; but, interestingly, in 45-day-old rats, L-NA given with or without EtOH lead to a significant fall in LHRH gene expression. Our findings indicate that the acute suppressive effects of EtOH on the hypothalamic-pituitary-gonadal axis of the purbertal male rat can be at least partially prevented by NO synthase blockade.