IS 10 mRNA stability and steady state levels in Escherichia coli: indirect effects of translation and role of rne function

AbstractGlucocorticoids (GC) are widely used clinically, despite the presence of significant side effects, including glucocorticoid-induced osteoporosis (GIOP). While GC are believed to act directly on osteoblasts and osteoclasts to promote osteoporosis, the detailed underlying molecular mechanism of GC-induced osteoporosis is still not fully elucidated. Here, we show that lymphocytes play a pivotal role in regulating GC-induced osteoporosis. We show that GIOP could not be induced in SCID mice that lack T cells, but it could be re-established by adoptive transfer of splenic T cells from wild-type mice. As expected, T cells in the periphery are greatly reduced by GC; instead, they accumulate in the bone marrow where they are protected from GC-induced apoptosis. These bone marrow T cells in GC-treated mice express high steady-state levels of NF-κB receptor activator ligand (RANKL), which promotes the formation and maturation of osteoclasts and induces osteoporosis. Taken together, these findings reveal a critical role for T cells in GIOP.

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Role of myosin phosphorylation in contractility of a platelet aggregate

AJP Cell Physiology ◽

10.1152/ajpcell.1985.249.3.c297 ◽

1985 ◽

Vol 249 (3) ◽

pp. C297-C303 ◽

Cited By ~ 9

Author(s):

M. E. Bromberg ◽

R. W. Sevy ◽

J. L. Daniel ◽

L. Salganicoff

Keyword(s):

Steady State ◽

Light Chain ◽

Myosin Phosphorylation ◽

Nonmuscle Cells ◽

Platelet Aggregate ◽

Steady State Levels ◽

State Contraction ◽

The Relationship ◽

Dose Dependent

The relationship between tension and myosin 20,000-Da light chain phosphorylation in intact nonmuscle cells was investigated using a preparation of thrombin-activated, irreversibly aggregated platelets known as the platelet strip. Steady-state levels of tension generated by the platelet strip were found to be linearly related to the level of myosin phosphorylation. This relationship was observed during dose-dependent relaxation induced by the adenylate cyclase activators prostaglandin (PG) E1 and PGI2, and during contraction induced by ADP, epinephrine, and the prostaglandin endoperoxide analogue U-46619, which did not appreciably alter the basal level of adenosine 3',5'-cyclic monophosphate in the preparation. The fully relaxed platelet strip, in the absence of external Ca2+, was associated with a level of 12% light chain phosphorylation, which increased to 72% on maximal contraction. During both relaxation and contraction, changes in myosin phosphorylation were also found to precede or coincide with tension changes. Furthermore, steady-state contraction induced by ADP was associated with a maintained elevation in the level of myosin phosphorylation. These results support the concept that myosin phosphorylation is an important regulatory mechanism for contractility in platelets.

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Role of Human Sec63 in Modulating the Steady-State Levels of Multi-Spanning Membrane Proteins

PLoS ONE ◽

10.1371/journal.pone.0049243 ◽

2012 ◽

Vol 7 (11) ◽

pp. e49243 ◽

Cited By ~ 13

Author(s):

Andreas Mades ◽

Katherina Gotthardt ◽

Karin Awe ◽

Jens Stieler ◽

Tatjana Döring ◽

...

Keyword(s):

Steady State ◽

Membrane Proteins ◽

Steady State Levels

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Glucocorticoid regulation of human pulmonary surfactant protein-B (SP-B) mRNA stability is independent of activated glucocorticoid receptor

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00420.2010 ◽

2011 ◽

Vol 300 (6) ◽

pp. L940-L950 ◽

Cited By ~ 10

Author(s):

Ceá C. Tillis ◽

Helen W. Huang ◽

Weizhen Bi ◽

Su Pan ◽

Shirley R. Bruce ◽

...

Keyword(s):

Steady State ◽

Glucocorticoid Receptor ◽

Mrna Stability ◽

Pulmonary Surfactant ◽

Surfactant Protein ◽

Airway Epithelial Cells ◽

Mrna Levels ◽

Airway Epithelial ◽

Surfactant Protein B ◽

Steady State Levels

Adequate expression of surfactant protein-B (SP-B) is critical in the function of pulmonary surfactant to reduce alveolar surface tension. Expression of SP-B mRNA is restricted to specific lung-airway epithelial cells, and human SP-B mRNA stability is increased in the presence of the synthetic glucocorticoid dexamethasone (DEX). Although the mechanism of SP-B mRNA stabilization by DEX is unknown, studies suggest involvement of the glucocorticoid receptor (GR). We developed a dual-cistronic plasmid-based expression assay in which steady-state levels of SP-B mRNA, determined by Northern analysis, reproducibly reflect changes in SP-B mRNA stability. Using this assay, we found that steady-state levels of SP-B mRNA increased greater than twofold in transfected human-airway epithelial cells (A549) incubated with DEX (10−7 M). DEX-mediated changes in SP-B mRNA levels required the presence of the SP-B mRNA 3′-untranslated region but did not require ongoing protein synthesis. The effect of DEX on SP-B mRNA levels was dose dependent, with maximal effect at 10−7 M. DEX increased levels of SP-B mRNA in cells lacking GR, and the presence of the GR antagonist RU486 did not interfere with the effect of DEX. Surprisingly, other steroid hormones (progesterone, estradiol, and vitamin D; 10−7 M) significantly increased SP-B mRNA levels, suggesting a common pathway of steroid hormone action on SP-B mRNA stability. These results indicate that the effect of DEX to increase SP-B mRNA stability is independent of activated GR and suggests that the mechanism is mediated by posttranscriptional or nongenomic effects of glucocorticoids.

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Analysis of YfgL and YaeT Interactions through Bioinformatics, Mutagenesis, and Biochemistry

Journal of Bacteriology ◽

10.1128/jb.01477-07 ◽

2007 ◽

Vol 190 (5) ◽

pp. 1507-1517 ◽

Cited By ~ 77

Author(s):

Phu Vuong ◽

Drew Bennion ◽

Jeremy Mantei ◽

Danielle Frost ◽

Rajeev Misra

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Membrane Protein ◽

Direct Contact ◽

Mutational Analysis ◽

Cross Linking ◽

Conserved Residues ◽

Protein Biogenesis ◽

Steady State Levels

ABSTRACT In Escherichia coli, YaeT, together with four lipoproteins, YfgL, YfiO, NlpB, and SmpA, forms a complex that is essential for β-barrel outer membrane protein biogenesis. Data suggest that YfgL and YfiO make direct but independent physical contacts with YaeT. Whereas the YaeT-YfiO interaction needs NlpB and SmpA for complex stabilization, the YaeT-YfgL interaction does not. Using bioinformatics, genetics, and biochemical approaches, we have identified three residues, L173, L175, and R176, in the mature YfgL protein that are critical for both function and interactions with YaeT. A single substitution at any of these sites produces no phenotypic defect, but two or three simultaneous alterations produce mild or yfgL-null phenotypes, respectively. Interestingly, biochemical data show that all YfgL variants, including those with single substitutions, have weakened in vivo YaeT-YfgL interaction. These defects are not due to mislocalization or low steady-state levels of YfgL. Cysteine-directed cross-linking data show that the region encompassing L173, L175, and R176 makes direct contact with YaeT. Using the same genetic and biochemical strategies, it was found that altering residues D227 and D229 in another region of YfgL from E221 to D229 resulted in defective YaeT bindings. In contrast, mutational analysis of conserved residues V319 to H328 of YfgL shows that they are important for YfgL biogenesis but not YfgL-YaeT interactions. The five YfgL mutants defective in YaeT associations and the yfgL background were used to show that SurA binds to YaeT (or another complex member) without going through YfgL.

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Analysis of the contribution of changes in mRNA stability to the changes in steady-state levels of cyclin mRNA in the mammalian cell cycle

FEBS Journal ◽

10.1111/j.1742-4658.2005.04918.x ◽

2005 ◽

Vol 272 (20) ◽

pp. 5217-5229 ◽

Cited By ~ 6

Author(s):

Anna Penelova ◽

Larry Richman ◽

Barbara Neupert ◽

Viesturs Simanis ◽

Lukas C. Kuhn

Keyword(s):

Cell Cycle ◽

Steady State ◽

Mammalian Cell ◽

Mrna Stability ◽

Mammalian Cell Cycle ◽

Steady State Levels

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Role of CheB and CheR in the Complex Chemotactic and Aerotactic Pathway of Azospirillum brasilense

Journal of Bacteriology ◽

10.1128/jb.00267-06 ◽

2006 ◽

Vol 188 (13) ◽

pp. 4759-4768 ◽

Cited By ~ 31

Author(s):

Bonnie B. Stephens ◽

Star N. Loar ◽

Gladys Alexandre

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Azospirillum Brasilense ◽

Model Organism ◽

Spatial Gradient ◽

Wild Type ◽

Temporal Gradient ◽

E Coli ◽

Significant Difference

ABSTRACT It has previously been reported that the alpha-proteobacterium Azospirillum brasilense undergoes methylation-independent chemotaxis; however, a recent study revealed cheB and cheR genes in this organism. We have constructed cheB, cheR, and cheBR mutants of A. brasilense and determined that the CheB and CheR proteins under study significantly influence chemotaxis and aerotaxis but are not essential for these behaviors to occur. First, we found that although cells lacking CheB, CheR, or both were no longer capable of responding to the addition of most chemoattractants in a temporal gradient assay, they did show a chemotactic response (albeit reduced) in a spatial gradient assay. Second, in comparison to the wild type, cheB and cheR mutants under steady-state conditions exhibited an altered swimming bias, whereas the cheBR mutant and the che operon mutant did not. Third, cheB and cheR mutants were null for aerotaxis, whereas the cheBR mutant showed reduced aerotaxis. In contrast to the swimming bias for the model organism Escherichia coli, the swimming bias in A. brasilense cells was dependent on the carbon source present and cells released methanol upon addition of some attractants and upon removal of other attractants. In comparison to the wild type, the cheB, cheR, and cheBR mutants showed various altered patterns of methanol release upon exposure to attractants. This study reveals a significant difference between the chemotaxis adaptation system of A. brasilense and that of the model organism E. coli and suggests that multiple chemotaxis systems are present and contribute to chemotaxis and aerotaxis in A. brasilense.

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Different Processing of an mRNA Species inBacillus subtilis and Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.182.3.689-695.2000 ◽

2000 ◽

Vol 182 (3) ◽

pp. 689-695 ◽

Cited By ~ 8

Author(s):

Martin Persson ◽

Elisabeth Glatz ◽

Blanka Rutberg

Keyword(s):

Escherichia Coli ◽

Steady State ◽

Inverted Repeat ◽

Fusion Transcript ◽

Temperature Sensitive ◽

Rnase Iii ◽

Transcription Terminator ◽

E Coli ◽

Steady State Levels ◽

The Stability

ABSTRACT Expression of the Bacillus subtilis glpD gene, which encodes glycerol-3-phosphate (G3P) dehydrogenase, is controlled by termination or antitermination of transcription. The untranslated leader sequence of glpD contains an inverted repeat that gives rise to a transcription terminator. In the presence of G3P, the antiterminator protein GlpP binds toglpD leader mRNA and promotes readthrough of the terminator. Certain mutations in the inverted repeat of theglpD leader result in GlpP-independent, temperature-sensitive (TS) expression of glpD. The TS phenotype is due to temperature-dependent degradation of theglpD mRNA. In the presence of GlpP, theglpD mRNA is stabilized. glpDleader-lacZ fusions were integrated into the chromosomes ofB. subtilis and Escherichia coli. Determination of steady-state levels of fusion mRNA in B. subtilis showed that the stability of the fusion mRNA is determined by theglpD leader part. Comparison of steady-state levels and half-lives of glpD leader-lacZ fusion mRNA inB. subtilis and E. coli revealed significant differences. A glpD leader-lacZ fusion transcript that was unstable in B. subtilis was considerably more stable in E. coli. GlpP, which stabilizes the transcript in B. subtilis, did not affect its stability in E. coli. Primer extension analysis showed that theglpD leader-lacZ fusion transcript is processed differently in B. subtilis and in E. coli. The dominating cleavage site in E. coli was barely detectable in B. subtilis. This site was shown to be a target ofE. coli RNase III.

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Dopaminergic regulation of avian prolactin gene transcription

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0310185 ◽

2003 ◽

Vol 31 (1) ◽

pp. 185-196 ◽

Cited By ~ 29

Author(s):

A Al Kahtane ◽

Y Chaiseha ◽

M El Halawani

Keyword(s):

Gene Expression ◽

Steady State ◽

Gene Transcription ◽

Mrna Stability ◽

Anterior Pituitary ◽

Good Evidence ◽

Pituitary Cells ◽

Anterior Pituitary Cells ◽

Steady State Levels ◽

Prl Gene

It is well documented that prolactin (PRL) release and PRL gene expression in birds are controlled by the tonic stimulation of hypothalamic vasoactive intestinal peptide (VIP). However, there is good evidence that dopamine (DA) exerts both stimulatory (at the hypothalamic level) and inhibitory (at the pituitary level) effects on PRL secretion. The interactions between VIP and DA in the regulation of PRL gene transcription are not known. This study was designed to examine the effects of a D(2) DA receptor agonist (D(2)AG; R(-)-propylnorapomorphine HCl) on basal and VIP-stimulated PRL gene transcription rate, PRL mRNA steady-state levels, PRL mRNA stability and PRL release from cultured turkey anterior pituitary cells. The D(2)AG (10(-)(10) M) completely inhibited the stimulatory effect of VIP (10(-)(7) M) upon nascent PRL mRNA as determined utilizing a nuclear run-on transcription assay. To examine further the effect of the D(2)AG on PRL mRNA post-transcriptional events, anterior pituitary cells were treated with different concentrations of D(2)AG (10(-)(12)-10(-)(4) M). Semi-quantitative RT-PCR and RIA were performed to determine the levels of PRL mRNA and PRL content in the medium respectively. The results show that D(2)AG inhibited VIP-stimulated PRL mRNA steady-state levels as well as basal and VIP-stimulated PRL release, effects which were diminished by the D(2) DA receptor antagonist, S(-)-eticlopride HCl (10(-)(10) M). Actinomycin D (5 microg/ml), an inhibitor of mRNA synthesis, was used to assess the effect of D(2)AG on PRL mRNA stability in response to VIP. The stimulatory effect of VIP on PRL mRNA stability was completely negated by the D(2)AG (from a half-life of 53.0+/-2.3 h in VIP-treated cells to 25.5+/-1.6 h in D(2)AG+VIP-treated cells, P<or=0.05). These results support the hypothesis that VIP and DA play a major role in the regulation of PRL gene expression in avian species, at both the transcriptional and post-transcriptional levels. In addition, these findings suggest that the DAergic system inhibits PRL release and synthesis by antagonizing VIP at the pituitary level via D(2) DA receptors.

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Role of STE genes in the mating factor signaling pathway mediated by GPA1 in Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.8.9.3777 ◽

1988 ◽

Vol 8 (9) ◽

pp. 3777-3783 ◽

Cited By ~ 52

Author(s):

N Nakayama ◽

Y Kaziro ◽

K Arai ◽

K Matsumoto

Keyword(s):

Steady State ◽

Signaling Pathway ◽

State Level ◽

G1 Arrest ◽

Gene Products ◽

Mating Efficiency ◽

Protein Functions ◽

Steady State Levels ◽

Mating Factor

The ste mutants (ste2, ste4, ste5, ste7, ste11, and ste12) are insensitive to mating factors and are, therefore, sterile. Roles of the STE gene products in the GPA1-mediated mating factor signaling pathway were studied by using ste gpa1 double mutants. Mating efficiency of a ste2 mutant defective in the alpha-factor receptor increased 1,000-fold in a gpa1 background, while G1 arrest and aberrant morphology (shmoo) caused by gpa1 were not suppressed by ste2. Furthermore, the steady-state level of the FUS1 transcript, which normally increases in response to mating factors, was also elevated when the GPA1 function was impaired. These results suggest that the GPA1 protein functions downstream of the STE2 receptor. Conversely, the sterility of ste4, ste5, ste7, ste11, and ste12 mutants was not suppressed by gpa1, but the lethal phenotype of gpa1 was suppressed by these ste mutations. Northern (RNA) blotting analysis revealed that the ste7, ste11, and ste12 mutations caused reductions of 50 to 70% in the steady-state levels of the GPA1 transcript, while ste4 had a slight effect and ste5 had no effect. This implies that the suppression by ste7, ste11, and ste12 could be due to reduced syntheses of additional components, including an effector, and that suppression by ste4 and ste5 may result from direct effects on the signaling pathway. The STE4, STE5, STE7, STE11, and STE12 products, therefore, appear to specify components of the signal transduction machinery, directly or indirectly, which function together with or downstream of GPA1.

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