scholarly journals Up-regulation of Per1 expression by estradiol and progesterone in the rat uterus

2007 ◽  
Vol 194 (3) ◽  
pp. 511-519 ◽  
Author(s):  
Pei-Jian He ◽  
Masami Hirata ◽  
Nobuhiko Yamauchi ◽  
Masa-aki Hattori
Keyword(s):  
Stromal Cells ◽  
Clock Genes ◽  
Staining Intensity ◽  
Reproductive Organ ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Rat Uterus ◽  
Ovarian Steroids ◽  
Direct Regulation

It has been established that estrogen can alter circadian rhythms in behavior and endocrine physiology in rodents. The uterus is a reproductive organ that is critically dependent on regulation by ovarian steroids. Here, we examined the expression of Per1 in different compartments of the uterus, and explored whether the ovarian steroids could regulate Per1 expression employing ovariectomized rat uterus. RT-PCR analysis showed that Per1 was cyclically expressed in the uterus. As revealed by in situ hybridization, the staining intensity of Per1 mRNA was stronger at ZT 8 than at ZT 0 in the uterine luminal epithelium (LE), stroma (S), and myometrium (M) compartments, but was not changed in the glandular epithelium (GE). Both in situ hybridization and immunofluorescence analyses revealed that estradiol (E2) administration induced high expression of Per1 in the LE, GE, and M, and less expression in the S compartment. Progesterone (P4) treatment resulted in an obvious enhancement of Per1 expression in the LE, GE, and S, but unchanged in the M compartment. Furthermore, the E2- and P4-activated Per1 expression was significantly repressed by their respective antagonists, ICI182 780 and RU486. These findings were further supported by RT-PCR analysis of Per1 expression in cultured uterine stromal cells. Collectively, the present data indicate that E2 and P4 might be involved in modification of circadian rhythm via direct regulation of the expression of clock genes.

Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants

Development ◽  
1999 ◽  
Vol 126 (20) ◽  
pp. 4405-4419 ◽  
Author(s):  
R. Ruiz-Medrano ◽  
B. Xoconostle-Cazares ◽  
W.J. Lucas
Keyword(s):  
Higher Plants ◽  
Phloem Transport ◽  
The Body ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Long Distance ◽  
Rna Molecules ◽  
Long Distance Transport ◽  
Meristem Development

Direct support for the concept that RNA molecules circulate throughout the plant, via the phloem, is provided through the characterisation of mRNA from phloem sap of mature pumpkin (Cucurbita maxima) leaves and stems. One of these mRNAs, CmNACP, is a member of the NAC domain gene family, some of whose members have been shown to be involved in apical meristem development. In situ RT-PCR analysis revealed the presence of CmNACP RNA in the companion cell-sieve element complex of leaf, stem and root phloem. Longitudinal and transverse sections showed continuity of transcript distribution between meristems and sieve elements of the protophloem, suggesting CmNACP mRNA transport over long distances and accumulation in vegetative, root and floral meristems. In situ hybridization studies conducted on CmNACP confirmed the results obtained using in situ RT-PCR. Phloem transport of CmNACP mRNA was proved directly by heterograft studies between pumpkin and cucumber plants, in which CmNACP transcripts were shown to accumulate in cucumber scion phloem and apical tissues. Similar experiments were conducted with 7 additional phloem-related transcripts. Collectively, these studies established the existence of a system for the delivery of specific mRNA transcripts from the body of the plant to the shoot apex. These findings provide insight into the presence of a novel mechanism likely used by higher plants to integrate developmental and physiological processes on a whole-plant basis.

In Vivo Modulation of T Cell and Monocyte Function Following Infusion of Mesenchymal Stromal Cells (MSC)

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4739-4739
Author(s):  
Cristina Castilla-LLorente ◽  
Mineo Iwata ◽  
Marco Mielcarek ◽  
V. Kraig Abrams ◽  
Billanna Hwang ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Lymph Nodes ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Mononuclear Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Blood Mononuclear Cells ◽  
Post Infusion

Abstract Mesenchymal stromal cells (MSCs) expanded ex vivo from aspirated marrow, have been used clinically with variable success to facilitate repair of infarcted hearts, treat graft versus host disease, and facilitate marrow reconstitution after radiation damage. While it is now generally acknowledged that these benefits are not the result of engraftment and differentiation of MSC into the target tissues, the mechanism by which these beneficial effects are achieved is not clear. We hypothesize that MSCs mediate their effect by activating an endogenous cell population which in turn modulates the immune response and/or homes to damaged tissue and participates in repair. To begin to test this hypothesis immortalized and cloned populations of canine MSC were generated to provide a consistent product for in vivo testing. One line, designated DS-1, has been evaluated in vivo by infusion into two normal dogs. Blood samples were taken pre infusion, immediately following infusion and at 1, 6, 24, 48, 72, 96 hours, and 7, 14, 21, and 28 days post infusion. Following infusion there was no consistent change in the number of WBC, however by day 3 there was a marked decrease in the % of CD3+ cells expressing FOXP3 and TGFβ in the blood, which did not recover to pre-infusion levels during the period of observation. At autopsy there was an increased number of these cells in the lymph nodes and spleen, whereas there was an overall decrease in the number of TH1 cells in these tissues. Quantitative RT- PCR analysis of cDNA prepared from blood mononuclear cells indicated an upregulation in the expression of CD133, Tie-2, and MARCO between 1–24 hours post infusion, and an increase in LOX1/OLR1 between 2–4 days. However the % of monocytes and the expression levels of CD14, CD68, CD45, and CD105/Endoglin were constant at all time points. Samples taken at 6 hours, 4 and 7 days post infusion were also analyzed for the presence of DS-1 cells by PCR and in vitro out growth assays. Results indicated that the DS-1cells were detectable up to 6 hours post infusion, but not thereafter. Adherent cells grown from blood mononuclear cells at days 4 and 7, displayed macrophage and endothelial cell morphologies. RT-PCR analysis of these cultures detected expression of macrophage associated markers CD14+/CD68+/MARCO+/LOX1+, as well as endothelial cell associated markers CD34+/CD144/VECAD+. These data indicate that a single infusion of DS-1 cells results in activation of circulating monocytes and a shift of regulatory T cells from the periphery to lymph nodes and spleen which persists for at least 28 days. We speculate that these changes may contribute to the immunomodulatory effects reported for some preparations of MSC.

Limited engraftment capacity of bone marrow–derived mesenchymal cells following T-cell–depleted hematopoietic stem cell transplantation

Blood ◽  
2000 ◽  
Vol 96 (10) ◽  
pp. 3637-3643 ◽  
Author(s):  
Daniela Cilloni ◽  
Carmelo Carlo-Stella ◽  
Franca Falzetti ◽  
Gabriella Sammarelli ◽  
Ester Regazzi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
T Cell ◽  
Stromal Cells ◽  
Human Leukocyte ◽  
Mesenchymal Cells ◽  
Mixed Chimerism ◽  
Pcr Analysis ◽  
Hematopoietic Stem ◽  
Leukocyte Antigen

The engraftment capacity of bone marrow–derived mesenchymal cells was investigated in 41 patients who had received a sex-mismatched, T-cell–depleted allograft from human leukocyte antigen (HLA)–matched or –mismatched family donors. Polymerase chain reaction (PCR) analysis of the human androgen receptor (HUMARA) or the amelogenin genes was used to detect donor-derived mesenchymal cells. Only 14 marrow samples (34%) from 41 consenting patients generated a marrow stromal layer adequate for PCR analysis. Monocyte-macrophage contamination of marrow stromal layers was reduced below the levels of sensitivity of HUMARA and amelogenin assays (5% and 3%, respectively) by repeated trypsinizations and treatment with the leucyl-leucine (leu-leu) methyl ester. Patients who received allografts from 12 female donors were analyzed by means of the HUMARA assay, and in 5 of 12 cases a partial female origin of stromal cells was demonstrated. Two patients who received allografts from male donors were analyzed by amplifying the amelogenin gene, and in both cases a partial male origin of stromal cells was shown. Fluorescent in situ hybridization analysis using a Y probe confirmed the results of PCR analysis and demonstrated in 2 cases the existence of a mixed chimerism at the stromal cell level. There was no statistical difference detected between the dose of fibroblast progenitors (colony-forming unit–F [CFU-F]) infused to patients with donor- or host-derived stromal cells (1.18 ± 0.13 × 104/kg vs 1.19 ± 0.19 × 104/kg; P ≥ .97). In conclusion, marrow stromal progenitors reinfused in patients receiving a T-cell–depleted allograft have a limited capacity of reconstituting marrow mesenchymal cells.

scholarly journals The identification and characterization of a testis-specific cDNA during spermatogenesis

2006 ◽  
Vol 11 (1) ◽  
Author(s):  
Ying Chen ◽  
Jiarui Hu ◽  
Ping Song ◽  
Wuming Gong
Keyword(s):  
Experimental Validation ◽  
Rna Binding ◽  
Pcr Analysis ◽  
Splicing Factors ◽  
Rt Pcr ◽  
Rich Domain ◽  
Pachytene Spermatocytes ◽  
Identification And Characterization

AbstractUsing bioinformatics and experimental validation, we obtained a cDNA (named srsf) which was exclusively expressed in the mouse testes. RT-PCR analysis showed that srsf mRNA was not expressed in the gonad during the sex determination period or during embryogenesis. In developing mouse tests, srsf expression was first detected on post-natal day 10, reached its highest level on day 23, and then reduced to and remained at a moderate level throughout adulthood. In situ hybridization analysis demonstrated that srsf mRNA was expressed in pachytene spermatocytes and round spermatids in the testes. The predicted protein contains one RNA-binding domain (RBD) and a serine-arginine rich domain (RS), which are characterized by some splicing factors of SR family members. These findings indicate that srsf may play a role during spermatogenesis.

Molecular identification of Ca2+-activated K+ channels in parotid acinar cells

2003 ◽  
Vol 284 (2) ◽  
pp. C535-C546 ◽  
Author(s):  
Keith Nehrke ◽  
Claire C. Quinn ◽  
Ted Begenisich
Keyword(s):  
Single Channel ◽  
Acinar Cells ◽  
Pcr Analysis ◽  
Rt Pcr ◽  
Channel Conductance ◽  
Single Channel Conductance ◽  
Parotid Acinar Cells ◽  
Large Conductance Channel ◽  
Intracellular Ca

We used molecular biological and patch-clamp techniques to identify the Ca2+-activated K+ channel genes in mouse parotid acinar cells. Two types of K+ channels were activated by intracellular Ca2+ with single-channel conductance values of 22 and 140 pS (in 135 mM external K+), consistent with the intermediate and maxi-K classes of Ca2+-activated K+ channels, typified by the mIK1 ( Kcnn4) and mSlo ( Kcnma1) genes, respectively. The presence of mIK1 mRNA was established in acinar cells by in situ hybridization. The electrophysiological and pharmacological properties of heterologously expressed mIK1 channels matched those of the native current; thus the native, smaller conductance channel is likely derived from the mIK1 gene. We found that parotid acinar cells express a single, uncommon splice variant of the mSlo gene and that heterologously expressed channels of this Slo variant had a single-channel conductance indistinguishable from that of the native, large-conductance channel. However, the sensitivity of this expressed Slo variant to the scorpion toxin iberiotoxin was considerably different from that of the native current. RT-PCR analysis revealed the presence of two mSlo β-subunits ( Kcnmb1 and Kcnmb4) in parotid tissue. Comparison of the iberiotoxin sensitivity of the native current with that of parotid mSlo expressed with each β-subunit in isolation and measurements of the iberiotoxin sensitivity of currents in cells from β1 knockout mice suggest that parotid acinar cells contain approximately equal numbers of homotetrameric channel proteins from the parotid variant of the Slo gene and heteromeric proteins composed of the parotid Slo variant in combination with the β4-subunit.

scholarly journals Occurrence of Two Distinct Urotensin II-Related Peptides in Zebrafish Provides New Insight into the Evolutionary History of the Urotensin II Gene Family

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2330-2341 ◽  
Author(s):  
Caroline Parmentier ◽  
Emilie Hameury ◽  
Christophe Dubessy ◽  
Feng B. Quan ◽  
Damien Habert ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Close Contact ◽  
Ventral Side ◽  
Pcr Analysis ◽  
Urotensin Ii ◽  
Rt Pcr ◽  
History Of ◽  
The Brain ◽  
Insight Into

The urotensin II (UII) family is currently known to consist of two paralogous peptides, namely UII and UII-related peptide (URP). In contrast to UII, which has been identified in all vertebrate classes so far, URP has only been characterized in tetrapods. We report here the occurrence of two distinct URP genes in teleosts, which we have named URP1 and URP2. Synteny analysis revealed that teleost URP1 and URP2 genes and tetrapod URP genes represent three distinct paralog genes that, together with the UII gene, probably arose from the two rounds of tetraploidization, which took place early in vertebrate evolution. The absence of URP in fish indicates that the corresponding gene has been lost in the teleost lineage, whereas it is likely that both the URP1 and URP2 genes have been lost in the tetrapod lineage. Quantitative RT-PCR analysis revealed that the URP2 gene is mainly expressed in the spinal cord and the brain in adult zebrafish. In situ hybridization experiments showed that in zebrafish embryos, URP2 mRNA-containing cells are located in the floor plate of the neural tube. In adult, URP2-expressing cells occur in close contact with the ventral side of the ependymal canal along the whole spinal cord, whereas in the brain, they are located below the fourth ventricle. These URP-expressing cells may correspond to cerebrospinal fluid-contacting neurons. In conclusion, our study reveals the occurrence of four distinct UII paralogous systems in vertebrates that may exert distinct functions, both in tetrapods and teleosts.

scholarly journals Differential distribution of oestrogen receptor-alpha and -beta mRNAs in the female reproductive organ of rats as revealed by in situ hybridization

2000 ◽  
Vol 165 (1) ◽  
pp. 59-66 ◽  
Author(s):  
CN Mowa ◽  
T Iwanaga
Keyword(s):  
In Situ Hybridization ◽  
Stromal Cells ◽  
Oestrogen Receptor ◽  
Reproductive Tract ◽  
Reproductive Organ ◽  
Differential Distribution ◽  
Oestrogen Receptor Alpha ◽  
Female Reproductive Organ ◽  
Significant Expression

The cellular distribution of two oestrogen receptor (ER) subtypes, ERalpha and ERbeta mRNAs, was studied in the entire female reproductive organ of the rat using in situ hybridization. Expression of ERalpha and ERbeta mRNAs was predominant in the reproductive tract and ovary respectively. ERalpha mRNA had the most pronounced expression in epithelial cells and subepithelial stromal cells from the oviduct to the vagina, while in the ovary it was moderately detected in only the theca folliculi and interstitial glands. The oviduct showed a region-dependent expression of ERalpha mRNA: the isthmus had the most intense signals while the infundibulum revealed a low intensity of expression. Signals for ERbeta mRNA in the ovary were most intense in the granulosa cells of healthy follicles, whereas degenerating follicles lacked any significant expression. Less intense signals for ERbeta mRNA were localized in the theca folliculi and corpus luteum. Detectable levels of ERbeta mRNA were observed in the subepithelial stromal cells from the oviduct to the vagina. This study shows that the two ER subtypes are differentially expressed in cells and compartments of the reproductive organ, suggesting that the mediation of oestrogen action in these tissues may be accomplished through the respective predominant receptor.

Removal of Rev-erbα inhibition contributes to the prostaglandin G/H synthase 2 expression in rat endometrial stromal cells

2015 ◽  
Vol 308 (8) ◽  
pp. E650-E661 ◽  
Author(s):  
Keishiro Isayama ◽  
Lijia Zhao ◽  
Huatao Chen ◽  
Nobuhiko Yamauchi ◽  
Yasufumi Shigeyoshi ◽  
...  
Keyword(s):  
Stromal Cells ◽  
Chromatin Immunoprecipitation ◽  
Clock Genes ◽  
Pcr Analysis ◽  
Core Component ◽  
Endometrial Stromal Cells ◽  
Transcript Levels ◽  
Rhythmic Expression ◽  
Decidual Cells ◽  
Implantation Sites

The rhythmic expression of clock genes in the uterus is attenuated during decidualization. This study focused on Ptgs2, which is essential for decidualization, as a putative clock-controlled gene, and aimed to reveal the functions of clock genes in relation to Ptgs2 during decidualization. We compared the transcript levels of clock genes in the rat uterus on days 4.5 (D4.5) and 6.5 of pregnancy. The transcript levels of clock genes ( Per2, Bmal1, Rorα, and Rev-erbα) had decreased at implantation sites on day 6.5 (D6.5e) compared with those on D4.5, whereas Ptgs2 transcripts had increased on D6.5e. Similar observations of Rev-erbα and Ptgs2 were also obtained in the endometrium on D6.5e by immunohistochemistry. In the decidual cells induced by medroxyprogesterone and 2- O-dibutyryl-cAMP, the rhythmic expression levels of clock genes were attenuated, whereas Ptgs2 transcription was induced. These results indicate that decidualization causes the attenuation of clock genes and the induction of Ptgs2. Furthermore, in the experiment of Bmal1 siRNA, the rhythmic expression of clock genes and Ptgs2 was attenuated by the siRNA. Transcript levels of Ptgs2 and prostaglandin (PG)E2 production were increased by treatment with the Rev-erbα antagonist, suggesting the contribution of the nuclear receptor Rev-erbα to Ptgs2 expression. Moreover, Rev-erbα knockdown enhanced the induction of Ptgs2 transcription and PGE2 production by forskolin. Chromatin immunoprecipitation-PCR analysis revealed that Rev-erbα could directly bind to a proximal RORE site of Ptgs2. Collectively, this study demonstrates that the attenuation of the circadian clock, especially its core component Rev-erbα, contributes to the induction of Ptgs2 during decidualization.

Real-time quantitative RT-PCR analysis of human bone marrow stromal cells during osteogenic differentiation in vitro

2002 ◽  
Vol 85 (4) ◽  
pp. 737-746 ◽  
Author(s):  
Oliver Frank ◽  
Manuel Heim ◽  
Marcel Jakob ◽  
Andrea Barbero ◽  
Dirk Schäfer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Osteogenic Differentiation ◽  
Real Time ◽  
Stromal Cells ◽  
Bone Marrow Stromal Cells ◽  
Marrow Stromal Cells ◽  
Human Bone ◽  
Pcr Analysis ◽  
Rt Pcr
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