Stress mRNA metabolism in canavanine-treated chicken embryo cells

1984 ◽  
Vol 4 (8) ◽  
pp. 1534-1541
Author(s):  
C N White ◽  
L E Hightower
Keyword(s):  
Steady State ◽  
Stress Proteins ◽  
Animal Cell ◽  
Stress Protein ◽  
High Rate ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Mrna Levels ◽  
Molecular Weights

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.

scholarly journals Stress mRNA metabolism in canavanine-treated chicken embryo cells.

1984 ◽  
Vol 4 (8) ◽  
pp. 1534-1541 ◽  
Author(s):  
C N White ◽  
L E Hightower
Keyword(s):  
Steady State ◽  
Stress Proteins ◽  
Animal Cell ◽  
Stress Protein ◽  
High Rate ◽  
Rrna Synthesis ◽  
28S Rrna ◽  
Mrna Levels ◽  
Molecular Weights

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.

scholarly journals Expression of stress proteins and mitochondrial chaperonins in chronically stimulated skeletal muscle

1995 ◽  
Vol 311 (1) ◽  
pp. 119-123 ◽  
Author(s):  
O I Ornatsky ◽  
M K Connor ◽  
D A Hood
Keyword(s):  
Steady State ◽  
Protein Import ◽  
Stress Proteins ◽  
Contractile Activity ◽  
Stress Protein ◽  
Type I ◽  
Mrna Levels ◽  
Striated Muscles ◽  
Organelle Biogenesis ◽  
Wide Range

Molecular chaperones and cytosolic stress proteins are actively involved in the stabilization, import and refolding of precursor proteins into mitochondria. The purpose of the present study was to evaluate the relationship between mitochondrial content under steady-state conditions, and during the induction of organelle biogenesis, with the expression of stress proteins and mitochondrial chaperonins. A comparison of steady-state levels of mitochondrial enzyme activity [cytochrome c oxidase (CYTOX)] with chaperonin levels [the heat-shock protein HSP60, the glucose-regulated protein GRP75 (mtHSP70)] in striated muscles possessing a wide range of oxidative capacities revealed a proportional expression between the two. This relationship was disrupted by chronic contractile activity brought about by 10 days of 10 Hz stimulation of the tibialis anterior (TA) muscle, which induced 2.4-fold increases in CYTOX activity, but 3.2- and 9.3-fold increases in HSP60 and GRP75 respectively. The inducible stress protein HSP70i was detected at low levels in control TA muscle, and was increased 9.6-fold by chronic contractile activity, to values comparable with those found in the unstressed soleus muscle. This increase occurred in the absence of changes in type I MHC levels, indicating independent regulation of these genes. Despite the increases in HSP60 and HSP70i proteins, contractile activity did not alter their respective mRNA levels, illustrating post-transcriptional mechanisms of gene regulation during contractile activity. In contrast, the mRNA levels encoding the co-chaperonin CPN10 were increased 3.3-fold by contractile activity. Thus, the expression of individual mitochondrial chaperonins is independently regulated and uncoordinated. The extent of the induction of these stress proteins and chaperonins by contractile activity exceeded that of membrane enzymes (e.g. CYTOX). It remains to be determined whether this marked induction of proteins comprising part of the protein import machinery is beneficial for the translocation of enzyme precursors into the mitochondria during conditions of accelerated biogenesis.

RAP1 is required for BAS1/BAS2- and GCN4-dependent transcription of the yeast HIS4 gene

1991 ◽  
Vol 11 (7) ◽  
pp. 3642-3651 ◽  
Author(s):  
C Devlin ◽  
K Tice-Baldwin ◽  
D Shore ◽  
K T Arndt
Keyword(s):  
Steady State ◽  
Binding Site ◽  
Binding Sites ◽  
Binding Activity ◽  
Micrococcal Nuclease ◽  
Mrna Levels ◽  
High Affinity ◽  
Steady State Levels

The major in vitro binding activity to the Saccharomyces cerevisiae HIS4 promoter is due to the RAP1 protein. In the absence of GCN4, BAS1, and BAS2, the RAP1 protein binds to the HIS4 promoter in vivo but cannot efficiently stimulate HIS4 transcription. RAP1, which binds adjacently to BAS2 on the HIS4 promoter, is required for BAS1/BAS2-dependent activation of HIS4 basal-level transcription. In addition, the RAP1-binding site overlaps with the single high-affinity HIS4 GCN4-binding site. Even though RAP1 and GCN4 bind competitively in vitro, RAP1 is required in vivo for (i) the normal steady-state levels of GCN4-dependent HIS4 transcription under nonstarvation conditions and (ii) the rapid increase in GCN4-dependent steady-state HIS4 mRNA levels following amino acid starvation. The presence of the RAP1-binding site in the HIS4 promoter causes a dramatic increase in the micrococcal nuclease sensitivity of two adjacent regions within HIS4 chromatin: one region contains the high-affinity GCN4-binding site, and the other region contains the BAS1- and BAS2-binding sites. These results suggest that RAP1 functions at HIS4 by increasing the accessibility of GCN4, BAS1, and BAS2 to their respective binding sites when these sites are present within chromatin.

scholarly journals Characterization of the Stringent Response and relBbu Expression in Borrelia burgdorferi

2003 ◽  
Vol 185 (3) ◽  
pp. 957-965 ◽  
Author(s):  
Julia Bugrysheva ◽  
Elena Y. Dobrikova ◽  
Marina L. Sartakova ◽  
Melissa J. Caimano ◽  
Thomas J. Daniels ◽  
...  
Keyword(s):  
Growth Rate ◽  
16S Rrna ◽  
Borrelia Burgdorferi ◽  
Stringent Response ◽  
Growth Conditions ◽  
Start Codon ◽  
Rrna Synthesis ◽  
Sequencing Analysis ◽  
Mrna Levels

ABSTRACT The stringent response is a global bacterial response to nutritional stress mediated by (p)ppGpp. We previously found that both noninfectious Borrelia burgdorferi strain B31 and infectious B. burgdorferi strain N40 produced large amounts of (p)ppGpp during growth in BSK-H medium and suggested that the stringent response was triggered in B. burgdorferi under these conditions. Here we report that (p)ppGpp levels in B. burgdorferi growing in BSK-II or BSK-H medium are not further increased by nutrient limitation or by serine hydroxamate-induced inhibition of protein synthesis and that the presence of (p)ppGpp during growth of N40 in BSK-H medium is not associated with decreased 16S rRNA synthesis. Decreased 16S rRNA synthesis was associated with the decreased growth rate of N40 seen during coculture with tick cells, which are growth conditions that were previously shown to decrease (p)ppGpp levels. One-half as much of the mRNA of the gene encoding the Rel protein of B. burgdorferi (relBbu ) was produced by B31 as by N40 during in vitro growth (2 ± 0.5 and 4 ± 0.8 fg of relBbu mRNA/ng of total Borrelia RNA, respectively). Although the amounts of N40 relBbu mRNA were identical during growth in vitro and in rat peritoneal chambers, they were markedly decreased during growth in nymphal ticks. In contrast to the lack of change in relBbu mRNA levels, larger amounts of a 78-kDa protein that was cross-reactive with antibodies to Bacillus subtilis RelBsu were detected in immunoblots of N40 lysates after growth in rat peritoneal chambers than after growth in vitro. Differences in the level of production of (p)ppGpp between B31 and N40 could not be explained by differences in relBbu promoters since identical transcriptional start sites 309 nucleotides upstream from the B31 and N40 relBbu ATG start codon and identical σ70-like promoters were identified by primer extension and sequencing analysis. relBbu complemented an Escherichia coli CF1693 relA spoT double mutant for growth on M9 minimal medium, and the transformed cells produced relBbu mRNA. These results indicate that relBbu is functional and that its transcription and translation and production of (p)ppGpp are affected by environmental conditions in strains N40 and B31. They also suggest that in B. burgdorferi, an organism with few rRNA operons that grows slowly, the role of (p)ppGpp may differ from the classic role played by this molecule in E. coli and that (p)ppGpp may not be responsible for growth rate control.

Regulation of steady-state follistatin mRNA levels in rat granulosa cells in vitro

1992 ◽  
Vol 9 (2) ◽  
pp. 147-156 ◽  
Author(s):  
U. Michel ◽  
J. W. McMaster ◽  
J. K. Findlay
Keyword(s):  
Steady State ◽  
Growth Factor ◽  
Epidermal Growth Factor ◽  
Cell Cultures ◽  
Granulosa Cells ◽  
Internal Standard ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Epidermal Growth

ABSTRACT The regulation of steady-state follistatin mRNA levels by different pituitary hormones and peptide factors was examined in granulosa cell cultures derived from diethylstilboestrol-treated immature rats. Cytosolic RNA from cell cultures was prepared by lysis and equal amounts of RNA from all samples were analysed with a solution—hybridization assay using a 32P-labelled antisense probe corresponding to a part of exon 5 together with a part of the 5′ end of exon 6 of the rat follistatin gene. In addition, a specific 35S-labelled probe for cyclophilin was used as an internal standard. The results show that 5 μg FSH/1 for 24 to 72 h stimulated steady-state follistatin mRNA levels, reaching levels 18·5-fold higher than controls. LH (0·2-100 μg/l) had only minor effects on follistatin mRNA levels in FSH-primed granulosa cells and prolactin, GH and IGF-I did not show any significant effects. Activin raised basal as well as FSH-stimulated steady-state follistatin mRNA levels up to ten- and twofold above controls respectively, whereas epidermal growth factor was found to inhibit FSH-stimulated follistatin mRNA levels in a dose-dependent manner. It is concluded that follistatin mRNA levels in granulosa cells are regulated by FSH rather than LH, and that the stimulation by FSH can be inhibited by epidermal growth factor but enhanced by activin. Activin alone was also capable of stimulating follistatin mRNA.

The biphasic modulation of inhibin mRNA levels and secretion by PMSG in rat granulosa cells in vitro

1991 ◽  
Vol 3 (2) ◽  
pp. 215 ◽  
Author(s):  
U Michel ◽  
Z Krozowski ◽  
J McMaster ◽  
JH Yu ◽  
JK Findlay
Keyword(s):  
Steady State ◽  
Granulosa Cells ◽  
Mrna Level ◽  
High Dose ◽  
Mrna Levels ◽  
High Concentration ◽  
A Subunit ◽  
High Doses ◽  
Vitro Effect

Granulosa cell cultures derived from diethylstilboestrol-treated immature rats were used to study the in vitro effect of pregnant mare serum gonadotrophin (PMSG) on steady state mRNA levels for the inhibin alpha and beta A subunits and the secretion of immunoreactive inhibin and progesterone. After 48 h treatment the dose-response curve of PMSG revealed a maximum stimulation (2.5-3.5 fold) of cytosolic alpha and beta A mRNAs over the range of 1 to 10 mU PMSG mL-1, with corresponding stimulation of inhibin secretion. A high dose of PMSG (160-500 mU mL-1) clearly suppressed inhibin alpha mRNA levels as well as inhibin secretion, whereas progesterone (P) was maximally stimulated (up to 600 fold). Although the level of cytosolic inhibin beta A subunit mRNA was also down-regulated by a high concentration of PMSG in the culture medium, the doses required to suppress its mRNA level to less than those of the control varied. These data demonstrate that low doses of follicle stimulating hormone/luteinizing hormone (FSH/LH)-like (PMSG) activity enhances and high doses decrease the steady-state mRNA levels of inhibin in rat granulosa cells in vitro; this biphasic regulation in vitro reflects the differential regulation of inhibin secretion observed during the rat oestrous cycle.

Complex transcriptional regulation of myc family gene expression in the developing mouse brain and liver

1991 ◽  
Vol 11 (12) ◽  
pp. 6007-6015
Author(s):  
L Xu ◽  
S D Morgenbesser ◽  
R A DePinho
Keyword(s):  
Gene Expression ◽  
Transcriptional Regulation ◽  
Steady State ◽  
High Rate ◽  
Developmental Expression ◽  
Tissue Type ◽  
Mrna Levels ◽  
Postnatal Brain ◽  
Transcriptional Initiation ◽  
Family Gene

myc family genes (c-, N-, and L-myc) have been shown to be differentially expressed with respect to tissue type and developmental stage. To define and compare the regulatory mechanisms governing their differential developmental expression, we examined the transcriptional regulation of each myc family member during murine postnatal brain and liver development. Nuclear run-on transcription assays demonstrated that both the rate of transcriptional initiation and the degree of transcriptional blocking contribute in a complex manner to the regulation of all three genes. During postnatal brain development, the relative contribution of each transcriptional control mechanism to the regulation of myc family gene expression was found to be different for each gene. For instance, while modulation of transcriptional attenuation did not appear to contribute to the down-regulation of L-myc expression, attenuation was found to be the dominant mechanism by which steady-state N-myc mRNA levels were down-regulated. Different transcriptional strategies were found to be employed in newborn versus adult developing liver for repression of N- and L-myc expression. Undetectable steady-state N- and L-myc mRNA levels in newborn liver were associated with a very low rate of transcriptional initiation, whereas the lack of N- and L-myc expression at the adult stage was accompanied by a high rate of initiation and a striking degree of transcriptional attenuation. Transcriptional attenuation in the N-myc gene was found to map to a region encoding a potential stem-loop structure followed by a thymine tract within the first exon and was not dependent on the use of a specific transcriptional start site.

Bleomycin induces apoptosis in human alveolar macrophages

1995 ◽  
Vol 269 (3) ◽  
pp. L318-L325 ◽  
Author(s):  
R. F. Hamilton ◽  
L. Li ◽  
T. B. Felder ◽  
A. Holian
Keyword(s):  
Dna Fragmentation ◽  
Stress Proteins ◽  
Mrna Levels ◽  
Dna Ladder ◽  
Dna Labeling ◽  
In Vitro Effects ◽  
Shock Proteins

Bleomycin (BLM) is an effective antineoplastic drug; however, cumulative dosage is often associated with inflammation that can progress to pulmonary fibrosis. The mechanisms by which this occurs are not understood, but they have been proposed to involve the alveolar macrophage (AM). In this study, we examined the in vitro effects of BLM on human AM cytotoxicity and the role of heat shock proteins (HSP or stress proteins) in this process. Although BLM did not cause marked necrosis, it caused significant DNA fragmentation detected by in situ DNA labeling and confirmed by BLM-induced DNA ladder formation after 24 h. The DNA fragmentation was significantly blocked by 10 and 50 microM ZnCl2, suggesting that BLM was inducing apoptosis. BLM did not alter intracellular protooncogene bcl-2 or glutathione levels. However, BLM significantly (50%) blocked HSP-72 expression by 4 h during a mild heat stress (39.8 degrees C). This inhibition occurs without affecting mRNA levels (in situ hybridization) for HSP-72 or overall protein synthesis ([35S]methionine incorporation), suggesting that BLM is blocking the stress response relatively specifically and post-transcriptionally. In summary, these results suggest that BLM causes apoptosis in human AM in vitro that is preceded by the inhibition of HSP-72 induction that appears to be caused by a posttranscriptional mechanism.

scholarly journals Antiatherosclerotic effect of dehydrocorydaline on ApoE−/− mice: inhibition of macrophage inflammation

2021 ◽  
Author(s):  
Bin Wen ◽  
Yuan-ye Dang ◽  
Su-hua Wu ◽  
Yi-min Huang ◽  
Kong-yang Ma ◽  
...  
Keyword(s):  
Vascular Inflammation ◽  
Chinese Herb ◽  
Gene Clusters ◽  
High Rate ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Protein Levels ◽  
Atherosclerosis Development

AbstractDespite improvements in cardiovascular disease (CVD) outcomes by cholesterol-lowering statin therapy, the high rate of CVD is still a great concern worldwide. Dehydrocorydaline (DHC) is an alkaloidal compound isolated from the traditional Chinese herb Corydalis yanhusuo. Emerging evidence shows that DHC has anti-inflammatory and antithrombotic benefits, but whether DHC exerts any antiatherosclerotic effects remains unclear. Our study revealed that intraperitoneal (i.p.) injection of DHC in apolipoprotein E-deficient (ApoE−/−) mice not only inhibited atherosclerosis development but also improved aortic compliance and increased plaque stability. In addition, DHC attenuated systemic and vascular inflammation in ApoE−/− mice. As macrophage inflammation plays an essential role in the pathogenesis of atherosclerosis, we next examined the direct effects of DHC on bone marrow-derived macrophages (BMDMs) in vitro. Our RNA-seq data revealed that DHC dramatically decreased the levels of proinflammatory gene clusters. We verified that DHC significantly downregulated proinflammatory interleukin (IL)-1β and IL-18 mRNA levels in a time- and concentration-dependent manner. Furthermore, DHC decreased lipopolysaccharide (LPS)-induced inflammation in BMDMs, as evidenced by the reduced protein levels of CD80, iNOS, NLRP3, IL-1β, and IL-18. Importantly, DHC attenuated LPS-induced activation of p65 and the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Thus, we conclude that DHC ameliorates atherosclerosis in ApoE−/− mice by inhibiting inflammation, likely by targeting macrophage p65- and ERK1/2-mediated pathways.

scholarly journals Sequence and expression of chicken and mouse rsk: homologs of Xenopus laevis ribosomal S6 kinase.

1989 ◽  
Vol 9 (9) ◽  
pp. 3850-3859 ◽  
Author(s):  
D A Alcorta ◽  
C M Crews ◽  
L J Sweet ◽  
L Bankston ◽  
S W Jones ◽  
...  
Keyword(s):  
Amino Acids ◽  
Xenopus Laevis ◽  
In Vitro Transcription ◽  
Catalytic Subunit ◽  
Mrna Levels ◽  
S6 Kinase ◽  
Molecular Weights ◽  
Mouse Cdna ◽  
Ribosomal S6 Kinase

We have previously reported the isolation of cDNAs encoding two closely related Xenopus ribosomal S6 kinases, S6KII alpha and -beta (S. W. Jones, E. Erikson, J. Blenis, J. L. Maller, and R. L. Erikson, Proc. Natl. Acad. Sci. USA 85:3377-3381, 1988). We report here the molecular cloning of one chicken and two mouse homologs of the Xenopus laevis cDNAs. As described for the Xenopus proteins, these cDNAs were found to predict polypeptides that contain two distinct kinase domains, of which one is most closely related to the catalytic subunit of cyclic AMP-dependent protein kinase and the other is most closely related to the catalytic subunit of phosphorylase b kinase. The three predicted proteins were more than 79% identical to the Xenopus S6KII alpha protein. The chicken and one of the mouse cDNAs were, respectively, 3.7 and 3.1 kilobase pairs in length, predicted proteins of 752 and 724 amino acids with molecular weights of 84.4 and 81.6 kilodaltons, and hybridized to mRNAs in fibroblasts and tissues of approximately 3.6 and 3.4 kilobases (kb). The second mouse cDNA was approximately 6.1 kilobase pairs and was not full length but predicted the C-terminal 633 amino acids of a protein that is similar to the C-terminal portion of Xenopus S6KII alpha. This clone hybridized to mRNA transcripts of 7.6 and 3.4 kb. In vitro transcription and translation of the chicken and the mouse cDNAs that predict complete proteins produced major products with apparent molecular weights of 96 and 84 kilodaltons. Analysis of mRNA levels in chicken tissues showed significant quantities of the 3.6-kb transcript in small and large intestine, spleen, and bursa. Both mouse cDNA were similarly expressed at significant levels in intestine, thymus, and lung; however, the 7.6-kb mRNA was differentially and more highly expressed in heart and brain. The two mouse cDNAs represent two different S6 kinase genes, as shown by comparison of their protein sequences, mRNA transcript sizes, genomic organizations, and nucleic acid sequences. We propose that this family of genes be named rsk, for ribosomal S6 kinase.

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