Overaccumulation of the chloroplast antisense RNA AS5 is correlated with decreased abundance of 5S rRNA in vivo and inefficient 5S rRNA maturation in vitro

α-Amanitin acts in vitro and in vivo as a selective inhibitor of nucleoplasmic RNA polymerases. Treatment of mice with low doses of α-amanitin causes the following changes in the synthesis, maturation and nucleocytoplasmic transfer of liver RNA species. 1. The synthesis of the nuclear precursor of mRNA is strongly inhibited and all electrophoretic components are randomly affected. The labelling of cytoplasmic mRNA is blocked. These effects may be correlated with the rapid and lasting inhibition of nucleoplasmic RNA polymerase. 2. The synthesis and maturation of the nuclear precursor of rRNA is inhibited within 30min. (a) The initial effect is a strong (about 80%) inhibition of the early steps of 45S precursor rRNA maturation. (b) The synthesis of 45S precursor rRNA is also inhibited and the effect increases from about 30% at 30min to more than 70% at 150min. (c) The labelling of nuclear and cytoplasmic 28S and 18S rRNA is almost completely blocked. The labelling of nuclear 5S rRNA is inhibited by about 50%, but that of cytoplasmic 5S rRNA is blocked. (d) The action of α-amanitin on the synthesis of precursor rRNA cannot be correlated with the slight gradual decrease of nucleolar RNA polymerase activity (only 10–20% inhibition at 150min). (e) The inhibition of precursor rRNA maturation and synthesis precedes the ultrastructural lesions of the nucleolus detected by standard electron microscopy. 3. The synthesis of nuclear 4.6S precursor of tRNA is not affected by α-amanitin. However, the labelling of nuclear and cytoplasmic tRNA is decreased by about 50%, which indicates an inhibition of precursor tRNA maturation. The results of this study suggest that the synthesis and maturation of the precursor of rRNA and the maturation of the precursor of tRNA are under the control of nucleoplasmic gene products. The regulator molecules may be either RNA or proteins with exceedingly fast turnover.

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In vitro processing at the 3'-terminal region of pre-18S rRNA by a nucleolar endoribonuclease.

Molecular and Cellular Biology ◽

10.1128/mcb.10.8.3868 ◽

1990 ◽

Vol 10 (8) ◽

pp. 3868-3872 ◽

Cited By ~ 15

Author(s):

C M Shumard ◽

C Torres ◽

D C Eichler

Keyword(s):

18S Rrna ◽

Precise Determination ◽

In Vivo Studies ◽

Rrna Sequence ◽

S1 Nuclease ◽

In Vitro Processing ◽

Rrna Maturation ◽

A Site

In an investigation of the possible involvement of a highly purified nucleolar endoribonuclease in processing of pre-rRNA at the 3' end of the 18S rRNA sequence, an in vitro synthesized pre-18S rRNA transcript containing the 3' end region of 18S rRNA and the 5' region of the first internal transcribed spacer (ITS1) was used as a substrate for the enzyme. Cleavages generated by the nucleolar RNase were localized by S1 nuclease protection analysis and by the direct release of labeled rRNA products. Precise determination of the specificity of cleavage was achieved by RNA sequence analysis with end-labeled rRNA transcripts. These data demonstrated that the purified nucleolar RNase cleaved the pre-18S rRNA transcript at three specific sites relative to the 3' region of 18S rRNA. The first two sites included the mature 3'-end 18S rRNA sequence and a site approximately 55 nucleotides downstream of the 3'-end 18S rRNA sequence, both of which corresponded directly to recent results (Raziuddin, R. D. Little, T. Labella, and D. Schlessinger, Mol. Cell. Biol. 9:1667-1671, 1989) obtained with transfected mouse rDNA in hamster cells. The other cleavage occurred approximately 35 nucleotides upstream from the mature 3' end in the 18S rRNA sequence. The results from this study mimic the results obtained from in vivo studies for processing in the 3' region of pre-18S rRNA, supporting the proposed involvement of this nucleolar endoribonuclease in rRNA maturation.

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P–442 Cancer does not adversely affect oocyte maturation in vitro with the exception of breast cancer

Human Reproduction ◽

10.1093/humrep/deab130.441 ◽

2021 ◽

Vol 36 (Supplement_1) ◽

Author(s):

I Viran. . Klun ◽

J Bedenk ◽

N Jancar

Keyword(s):

Breast Cancer ◽

Cancer Patients ◽

Oocyte Maturation ◽

In Vitro Maturation ◽

Hormonal Stimulation ◽

Infertile Women ◽

Maturation In Vitro ◽

Infertile Patients

Abstract Study question Do different types of cancer affect the success of oocyte maturation in vitro compared to infertile women included in the in vitro fertilization (IVF) program? Summary answer Cancer does not adversely affect oocyte maturation in vitro, with the exception of breast cancer, compared to infertile women in the in vitro fertilization program. What is known already Vitrification and storage of oocytes in liquid nitrogen is one of the real options for maintaining reproductive function in cancer patients. Despite careful hormonal stimulation of the ovaries, however, the proportion of oocytes is immature and lost to the patient. In vitro maturation of oocytes can play an important role in resolving immature oocytes and increasing the chances of conception in cancer patients. Moreover, it can mean a safe way to store oocytes when ovarian hormonal stimulation could worsen the disease. Therefore, the aim of this study was to determine whether different types of cancer affect oocyte in vitro maturation. Study design, size, duration After ovarian stimulation in 18 cancer patients, the number and maturity of oocytes were compared to 21 infertile patients in the IVF program over a three-year period. In both groups, 119 germinal vesicle-GV oocytes were matured in vitro to compare the maturation rate. After IVF in a subset of 17 infertile patients, the fertilization of in vitro and in vivo matured oocytes was compared in the same cycles. The procedure was considered in cancer patients. Participants/materials, setting, methods In this prospective study, forty-five GV oocytes in cancer patients and 74 GV oocytes in infertile patients underwent in vitro maturation procedure. Each oocyte was matured in vitro in the MediCult IVM System by conditioning in LAG medium and maturation for up to 28 hours in IVM medium with added hormones FSH and hCG, in coculture with cumulus cells from mature oocytes in the same patients. Oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Main results and the role of chance After controlled ovarian hormonal stimulation, 198 oocytes were retrieved in cancer patients and 259 oocytes in infertile women and there were no significant differences in the number of retrieved oocytes, proportion of degenerated oocytes and proportion of GV oocytes. In cancer patients, the proportion of oocytes that matured in vitro was lower than in infertile patients (66.0 vs. 80.0%), but the difference was not significant. Among cancer patients, the oocyte maturation rate tended to be lower in patients with breast cancer than in patients with other cancers (54.5% vs. 81.2%; difference not significant). However, in patients with breast cancer, significantly fewer oocytes matured in vitro than in infertile patients (54.5% vs. 80.0%; P < 0.05, Chi-Square test) even though they tended to be younger (29.3 ± 7.4 vs. 33.4 ± 5.0 years; non-significant difference). After in vitro maturation, there was a 13% increase in mature oocyte yield in cancer patients and a 20.1% increase in infertile women with no significant difference observed. After ICSI in a subset of infertile women, there was approximately the same fertilization rate between oocytes matured in vitro and in vivo (55.1% vs. 57.0%) in the same cycles. Limitations, reasons for caution For ICSI in oocytes matured in vitro, we had to use semen collected the day before, while oocytes matured in vivo were fertilized with fresh semen in the same cycle. Therefore, we could not compare the development of embryos in both groups. Wider implications of the findings: In vitro maturation of oocytes in connection with their vitrification or vitrification of embryos after their fertilization appears to be a valuable way to maintain the fertility of young cancer patients, but a worse outcome is expected in breast cancer patients. Trial registration number National Medical Ethical Committee Approval, No. 0120–222/2016–2; KME 115/04/16.

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Maturation of human oocytes in vitro and their developmental competence

Reproduction ◽

10.1530/rep.0.1210051 ◽

2001 ◽

pp. 51-75 ◽

Cited By ~ 221

Author(s):

A Trounson ◽

C Anderiesz ◽

G Jones

Keyword(s):

Embryo Development ◽

Oocyte Maturation ◽

Growth Phase ◽

Developmental Competence ◽

Minimum Size ◽

Success Rates ◽

Human Oocytes ◽

Maturation In Vitro

Complete maturation of oocytes is essential for the developmental competence of embryos. Any interventions in the growth phase of the oocyte and the follicle in the ovary will affect oocyte maturation, fertilization and subsequent embryo development. Oocyte size is associated with maturation and embryo development in most species examined and this may indicate that a certain size is necessary to initiate the molecular cascade of normal nuclear and cytoplasmic maturation. The minimum size of follicle required for developmental competence in humans is 5-7 mm in diameter. Maturation in vitro can be accomplished in humans, but is associated with a loss of developmental competence unless the oocyte is near completion of its preovulatory growth phase. This loss of developmental competence is associated with the absence of specific proteins in oocytes cultured to metaphase II in vitro. The composition of culture medium used successfully for maturation of human oocytes is surprisingly similar to that originally developed for maturation of oocytes in follicle culture in vitro. The presence of follicle support cells in culture is necessary for the gonadotrophin-mediated response required to mature oocytes in vitro. Gonadotrophin concentration and the sequence of FSH and FSH-LH exposure may be important for human oocytes, particularly those not exposed to the gonadotrophin surge in vivo. More research is needed to describe the molecular and cellular events, the presence of checkpoints and the role of gene expression, translation and protein uptake on completing oocyte maturation in vitro and in vivo. In the meantime, there are very clear applications for maturing oocytes in human reproductive medicine and the success rates achieved in some of these special applications are clinically valuable.

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Protein synthesis and phosphorylation during bovine oocyte maturation in vitro and in vivo,

Cell Differentiation and Development ◽

10.1016/0922-3371(89)90148-2 ◽

1989 ◽

Vol 27 ◽

pp. 39 ◽

Cited By ~ 1

Author(s):

P.M.M. Kastrop ◽

M.M. Bevers ◽

O.H.J. Destrée ◽

Th.A.M. Kruip

Keyword(s):

Protein Synthesis ◽

Oocyte Maturation ◽

Bovine Oocyte ◽

Maturation In Vitro

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Inhibition of hepatitis C virus gene expression by adenoviral vectors encoding antisense RNA in vitro and in vivo

Journal of Hepatology ◽

10.1016/j.jhep.2010.11.010 ◽

2011 ◽

Vol 55 (1) ◽

pp. 19-28 ◽

Cited By ~ 9

Author(s):

Maria A. González-Carmona ◽

Annabelle Vogt ◽

Thomas Heinicke ◽

Maria Quasdorff ◽

Per Hoffmann ◽

...

Keyword(s):

Gene Expression ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Antisense Rna ◽

Adenoviral Vectors ◽

Virus Gene ◽

Virus Gene Expression

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Poorly crystalline apatites: evolution and maturation in vitro and in vivo

Journal of Bone and Mineral Metabolism ◽

10.1007/s00774-004-0488-0 ◽

2004 ◽

Vol 22 (4) ◽

Cited By ~ 89

Author(s):

Sophie Cazalbou ◽

Christ�le Combes ◽

Diane Eichert ◽

Christian Rey ◽

Melvin J. Glimcher

Keyword(s):

Maturation In Vitro

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Leukemic blasts fail to differentiate into mature antigen presenting cells, and may hinder dendritic cell maturation in vitro and in vivo

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.10556 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 10556-10556

Author(s):

J. Rosenblatt ◽

R. Stone ◽

C. Lenahan ◽

Z. Wu ◽

B. Vasir ◽

...

Keyword(s):

T Cells ◽

Dendritic Cells ◽

Tumor Vaccine ◽

Leukemia Cells ◽

Phenotypic Characteristics ◽

Gm Csf ◽

Maturation In Vitro ◽

Dc Maturation

10556 Background: Dendritic cells (DC) play a key role in the development of tumor specific immune responses. Dendritic cells differentiated from leukemic blasts (LDC) are being explored as a tumor vaccine in AML. We examined the phenotypic and functional characteristics of LDC, the phenotypic characteristics of native DC in AML patients, and the effect of leukemic blasts on the phenotype of DC generated from normal donors. Methods: Leukemia blasts were isolated from peripheral blood of 24 patients with AML. LDC were generated by culturing blasts in the presence of GM-CSF, IL-4 and TNFa for 7 days. The phenotype of circulating DC1 (CD11C+/lin-) and DC2 (CD123+/ lin-) in AML patients was assessed by multichannel FACS analysis. To assess the effect of blasts on DC maturation, adherent mononuclear cells were isolated from normal donors, combined with leukemia cells in a 10:1 ratio, and cultured with GM-CSF, IL-4, and TNFa. Results: LDC demonstrate only modest expression of the costimulatory molecules CD80 and CD86 (mean expression 10% and 32%) and poorly express the maturation marker CD83 (mean expression 4%). Interferon gamma production by autologous T cells was not higher after stimulation with LDC than with blasts. LDC stimlation resulted in a 2 fold increase in both CD4+/CD25+/CD69+ (activated) and CD4+/CD25+/FOXP3+ (regulatory) T cells. Given the inability of leukemia progenitors to differentiate into phenotypically mature DC, we assessed whether leukemia cells directly inhibit differentiation of DC from normal progenitors. Expression of costimulatory molecules was decreased in DC differentiated in the presence of blasts. Mean expression of CD80, CD83, and CD86 was 16%, 2%, 83% and 49%, 10%, 99% for DCs generated in the presence or absence blasts respectively. Phenotypic characteristics of native DC in patients with AML were examined. In 3 experiments, a predominance of DC2 was seen (ratio DC2/DC1 5), and both DC1 and DC2 poorly expressed CD83 (mean expression 9% DC1, 0.9% DC2). Conclusions: LDC have phenotypic and functional deficiencies, limiting their efficacy as a tumor vaccine. Contact with leukemic blasts may inhibit DC maturation in vitro and in vivo, which may contribute to the lack of effective antitumor immunity in AML patients. No significant financial relationships to disclose.

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TACI-BLyS signaling via B-cell–dendritic cell cooperation is required for naive CD8+ T-cell priming in vivo

Blood ◽

10.1182/blood-2004-12-4708 ◽

2006 ◽

Vol 107 (2) ◽

pp. 594-601 ◽

Cited By ~ 31

Author(s):

Yaiza Diaz-de-Durana ◽

George T. Mantchev ◽

Richard J. Bram ◽

Alessandra Franco

Keyword(s):

B Cells ◽

Dendritic Cell ◽

B Cell ◽

B Lymphocyte ◽

Costimulatory Molecule ◽

Early Signal ◽

Maturation In Vitro ◽

Deficient Mice

AbstractWe demonstrated that B-cell–dendritic cell (DC) interactions via transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) and B-lymphocyte stimulator (BLyS) provide an early signal critical to generate adequate numbers of mature antigen presenting cells (APCs) to prime naive CD8+ T cells (CTLs) in vivo. Evidence that B cells are required for efficient CTL generation in mice and that reconstitution with wild-type but not TACI-knockout B cells restored normal CTL responses support our conclusion. Moreover, low doses of a TACI fusion protein (TACI-Fc) that express the extracellular domain of TACI (amino acid [aa] 1-126) restored CTL priming in B-cell–deficient mice in vivo and induced DC maturation in vitro. In fact, following interactions with B cells, splenic DCs rapidly express the CD86 costimulatory molecule, to an extent comparable to the exposure to antigenic stimuli. BLyShigh peptide-pulsed bone marrow–derived DCs, used as vaccines in vivo, cannot generate CTLs in B-cell–deficient and TACI-deficient mice, strongly supporting a need for B-cell–DC cooperation through TACI-BLyS during CTL first encounter with antigens in vivo.

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LncRNA PITPNA-AS1 as a Potential Diagnostic Marker and Therapeutic Target Promotes Hepatocellular Carcinoma Progression via Modulating miR-448/ROCK1 Axis

Frontiers in Medicine ◽

10.3389/fmed.2021.668787 ◽

2021 ◽

Vol 8 ◽

Author(s):

Qing-fang Wang ◽

Qing-lin Wang ◽

Ming-bo Cao

Keyword(s):

Hepatocellular Carcinoma ◽

Antisense Rna ◽

Diagnostic Marker ◽

Luciferase Assay ◽

Migration And Invasion ◽

Biological Functions ◽

Non Coding Rnas ◽

Hcc Cells

Background: Long non-coding RNAs are critical to hepatocellular carcinoma (HCC) developments. LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) is a new regulator in several tumors. However, the mechanism by which PITPNA-AS1 mediates the tumorigenesis of HCC remains unclear.Methods: RT-qPCR was used to detect the level of PITPNA-AS1 in HCC specimens and cells. The biological functions of PITPNA-AS1 were explored by several functional experiments in vivo and in vitro. The binding relationship among PITPNA-AS1, miR-448 and ROCK1 were studied by Luciferase assay and pull-down assays.Results: We found that PITPNA-AS1 expressions were distinctly upregulated in both HCC specimens and cell lines. High PITPNA-AS1 levels were an unfavorable biomarker for patients with HCC. Functionally, knockdown of PITPNA-AS1 suppressed the proliferation, migration and invasion of HCC cells. Mechanistically, PITPNA-AS1 functioned as competing endogenous RNA to increase ROCK1 expressions via sponging miR-448.Conclusion: The newly identified PITPNA-AS/miR-448/ROCK1 axis promoted the oncogenicity of HCC cells. This novel axis is likely to be a promising HCC therapeutic aim.

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