304. Murine HIF-1α localisation by immunohistochemistry in a mouse reproductive tissue

2005 ◽  
Vol 17 (9) ◽  
pp. 129
Author(s):  
E. Camp-Dotlic ◽  
D. Froiland ◽  
K. L. Kind ◽  
H. Irving-Rodgers ◽  
J. G. Thompson ◽  
...  
Keyword(s):  
Reproductive Function ◽  
Protein Detection ◽  
Staining Intensity ◽  
Principal Cells ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Mouse Tissues ◽  
Cervical Dislocation ◽  
The Stability

Hypoxia-inducible factors (HIFs) are heterodimeric transcription factors consisting of an α and β subunit. The level of O2 within cells regulates the stability of HIF-1α and HIF-1 is considered the primary mediator of cellular responses to hypoxia, helping restore O2 homeostasis by promoting the expression of hypoxia-sensitive genes involved in cell survival, angiogenesis, cell proliferation and metabolism. There are few published studies investigating the role of HIF-1 protein in mouse tissues using immunohistochemistry, due to the lack of a reliable protocol and the inability of many commercially available antibodies to detect murine HIF-1α protein. We have developed a protocol that has allowed us to analyse the presence and location of HIF-1α protein in the mouse epididymis and found that it was predominantly located in the nucleus of discrete principal cells in the epididymis of mice housed under normoxic conditions and sacrificed by cervical dislocation. Interestingly, a 2.5× increase (P < 0.05) of HIF-1α protein staining intensity in both the nucleus and cytoplasm of principal cells in the epididymis was detected in mice housed under normoxic conditions but sacrificed with CO2 gas, compared to mice sacrificed by cervical dislocation. HIF-1α protein detection was 3-fold increased in the nucleus and cytoplasm of principal cells when mice were exposed to hypoxic conditions (6% O2 for 1 h). Our results demonstrate that murine HIF-1α can be detected in discrete cells under normoxic conditions, suggesting local differences in O2. Acute hypoxic responses, via deliberate exposure or even CO2 euthanasia can significantly upregulate HIF-1α protein levels. Further studies will investigate the role of HIFs and hypoxia in male and female reproductive function.

scholarly journals cdc37 is essential for JNK pathway activation and wound closure in Drosophila

2019 ◽  
Vol 30 (21) ◽  
pp. 2651-2658
Author(s):  
Chan-wool Lee ◽  
Young-Chang Kwon ◽  
Youngbin Lee ◽  
Min-Yoon Park ◽  
Kwang-Min Choe
Keyword(s):  
Cell Growth ◽  
Wound Closure ◽  
Cell Growth And Migration ◽  
Jnk Pathway ◽  
Protein Levels ◽  
Pathway Activation ◽  
And Migration ◽  
The Stability ◽  
Jnk Activation

Wound closure in the Drosophila larval epidermis mainly involves nonproliferative, endocyling epithelial cells. Consequently, it is largely mediated by cell growth and migration. We discovered that both cell growth and migration in Drosophila require the cochaperone-encoding gene cdc37. Larvae lacking cdc37 in the epidermis failed to close wounds, and the cells of the epidermis failed to change cell shape and polarize. Likewise, wound-induced cell growth was significantly reduced, and correlated with a reduction in the size of the cell nucleus. The c-Jun N-terminal kinase (JNK) pathway, which is essential for wound closure, was not typically activated in injured cdc37 knockdown larvae. In addition, JNK, Hep, Mkk4, and Tak1 protein levels were reduced, consistent with previous reports showing that Cdc37 is important for the stability of various client kinases. Protein levels of the integrin β subunit and its wound-induced protein expression were also reduced, reflecting the disruption of JNK activation, which is crucial for expression of integrin β during wound closure. These results are consistent with a role of Cdc37 in maintaining the stability of the JNK pathway kinases, thus mediating cell growth and migration during Drosophila wound healing.

scholarly journals Opposing Roles of Leptin and Ghrelin in the Equine Corpus Luteum Regulation: AnIn VitroStudy

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
António Galvão ◽  
Angela Tramontano ◽  
Maria Rosa Rebordão ◽  
Ana Amaral ◽  
Pedro Pinto Bravo ◽  
...  
Keyword(s):  
Corpus Luteum ◽  
Reproductive Function ◽  
Secretory Activity ◽  
Dependent Manner ◽  
Angiogenic Activity ◽  
Metabolic Hormones ◽  
Protein Levels ◽  
Dose Dependent

Metabolic hormones have been associated with reproductive function modulation. Thus, the aim of this study was: (i) to characterize the immunolocalization, mRNA and protein levels of leptin (LEP), Ghrelin (GHR) and respective receptors LEPR and Ghr-R1A, throughout luteal phase; and (ii) to evaluate the role of LEP and GHR on progesterone (P4), prostaglandin (PG) E2and PGF2α, nitric oxide (nitrite), tumor necrosis factor-α(TNF); macrophage migration inhibitory factor (MIF) secretion, and on angiogenic activity (BAEC proliferation), in equine corpus luteum (CL) from early and mid-luteal stages. LEPR expression was decreased in late CL, while GHR/Ghr-R1A system was increased in the same stage. Regarding secretory activity, GHR decreased P4in early CL, but increased PGF2α, nitrite and TNF in mid CL. Conversely, LEP increased P4, PGE2, angiogenic activity, MIF, TNF and nitrite during early CL, in a dose-dependent manner. Thein vitroeffect of LEP on secretory activity was reverted by GHR, when both factors acted together. The present results evidence the presence of LEP and GHR systems in the equine CL. Moreover, we suggest that LEP and GHR play opposing roles in equine CL regulation, with LEP supporting luteal establishment and GHR promoting luteal regression. Finally, a dose-dependent luteotrophic effect of LEP was demonstrated.

scholarly journals An Essential Role of INI1/hSNF5 Chromatin Remodeling Protein in HIV-1 Posttranscriptional Events and Gag/Gag-Pol Stability

2016 ◽  
Vol 90 (21) ◽  
pp. 9889-9904 ◽  
Author(s):  
Annalena La Porte ◽  
Jennifer Cano ◽  
Xuhong Wu ◽  
Doyel Mitra ◽  
Ganjam V. Kalpana
Keyword(s):  
Particle Production ◽  
Protein Levels ◽  
Mediated Effects ◽  
Viral Particle Production ◽  
Novel Strategy ◽  
The Stability ◽  
Novel Therapeutic Strategies ◽  
Hiv 1 ◽  
Rna Levels

ABSTRACTINI1/hSNF5/SMARCB1/BAF47 is an HIV-specific integrase (IN)-binding protein that influences HIV-1 transcription and particle production. INI1 binds to SAP18 (Sin3a-associated protein, 18 kDa), and both INI1 and SAP18 are incorporated into HIV-1 virions. To determine the significance of INI1 and the INI1-SAP18 interaction during HIV-1 replication, we isolated a panel ofSAP18-interaction-defective (SID)-INI1 mutants using a yeast reverse two-hybrid screen. The SID-INI1 mutants, which retained the ability to bind to IN, cMYC, and INI1 but were impaired for binding to SAP18, were tested for their effects on HIV-1 particle production. SID-INI1 dramatically reduced the intracellular Gag/Gag-Pol protein levels and, in addition, decreased viral particle production. The SID-INI1-mediated effects were less dramatic intranscomplementation assays using IN deletion mutant viruses with Vpr-reverse transcriptase (RT)-IN. SID-INI1 did not inhibit long-terminal-repeat (LTR)-mediated transcription, but it marginally decreased the steady-stategagRNA levels, suggesting a posttranscriptional effect. Pulse-chase analysis indicated that in SID-INI1-expressing cells, the pr55Gag levels decreased rapidly. RNA interference analysis indicated that small hairpin RNA (shRNA)-mediated knockdown ofINI1reduced the intracellular Gag/Gag-Pol levels and further inhibited HIV-1 particle production. These results suggest that SID-INI1 mutants inhibit multiple stages of posttranscriptional events of HIV-1 replication, including intracellular Gag/Gag-Pol RNA and protein levels, which in turn inhibits assembly and particle production. Interfering INI1 leads to a decrease in particle production and Gag/Gag-Pol protein levels. Understanding the role of INI1 and SAP18 in HIV-1 replication is likely to provide novel insight into the stability of Gag/Gag-Pol, which may lead to the development of novel therapeutic strategies to inhibit HIV-1 late events.IMPORTANCESignificant gaps exist in our current understanding of the mechanisms and host factors that influence HIV-1 posttranscriptional events, includinggagRNA levels, Gag/Gag-Pol protein levels, assembly, and particle production. Our previous studies suggested that the IN-binding host factor INI1 plays a role in HIV-1 assembly. An ectopically expressed minimal IN-binding domain of INI1, S6, potently and selectively inhibited HIV-1 Gag/Gag-Pol trafficking and particle production. However, whether or not endogenous INI1 and its interacting partners, such as SAP18, are required for late events was unknown. Here, we report that endogenous INI1 and its interaction with SAP18 are necessary to maintain intracellular levels of Gag/Gag-Pol and for particle production. Interfering INI1 or the INI1-SAP18 interaction leads to the impairment of these processes, suggesting a novel strategy for inhibiting posttranscriptional events of HIV-1 replication.

scholarly journals Role of Hakai in m6A modification pathway in Drosophila

2020 ◽  
Author(s):  
Yanhua Wang ◽  
Lifeng Zhang ◽  
Hang Ren ◽  
Jian Guo ◽  
Decai Mao ◽  
...  
Keyword(s):  
Nuclear Protein ◽  
Rna Degradation ◽  
Start Codon ◽  
Main Function ◽  
Protein Levels ◽  
Wing Discs ◽  
The Stability ◽  
And Behavior ◽  
Mammalian System

Abstract N6-methyladenosine (m6A), the most abundant internal modification in eukaryotic mRNA, is deposited by a multi-component writer complex. Hakai is an E3 ubiquitin ligase that interacts with several m6A writer subunits in proteomic studies, however, its role in m6A methylation in animals has not been systematically characterized. Here we show that Hakai colocalizes and interacts with other m6A writer components in Drosophila, and Hakai mutants exhibit typic m6A pathway phenotypes, such as lowered m6A levels in mRNA, aberrant Sxl alternative splicing, wing and behavior defects, common reduced m6A peaks and mis-regulated genes with Mettl3 and Mettl14 mutants. These results demonstrate that Hakai is a core component of the m6A writer complex comprised of seven conserved subunits. Interestingly, our stringent meRIP-seq experiments indicate that the effective m6A modification, which depends on the writer complex, is mostly distributed in 5’ UTR and near start codon in Drosophila, in contrast to the mammalian system. We define a set of high-confident m6A methylation sites in 5’ UTR in adult flies and it is unlikely the main function of m6A modification in Drosophila is through RNA degradation. Furthermore, we find that Hakai is required for the protein levels of other m6A writer components Fl(2)d and Flacc, but not Nito. Finally, Hakai does not mediate the stability of E-cadherin in wing discs, suggesting its major role as a nuclear protein.

scholarly journals Hypoxic Proliferation of Osteosarcoma Cells Depends on Arginase II

2016 ◽  
Vol 39 (2) ◽  
pp. 802-813 ◽  
Author(s):  
Bhuvana A. Setty ◽  
Yi Jin ◽  
Peter J. Houghton ◽  
Nicholas D. Yeager ◽  
Thomas G. Gross ◽  
...  
Keyword(s):  
Cellular Proliferation ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Small Interfering Rnas ◽  
Osteosarcoma Cells ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Human Osteosarcoma Cell ◽  
Arginase Ii

Background/Aims: Despite significant advancements in the diagnosis and treatment of osteosarcoma, the overall survival has remained relatively unchanged for over two decades. Hypoxic conditions have been demonstrated in solid tumors and are associated with increased cell proliferation and angiogenesis. L-arginine metabolism by arginase produces L-ornithine, the precursor for polyamine and proline synthesis required for cellular proliferation. We hypothesized that hypoxia would increase cellular proliferation via arginase induction in human osteosarcoma cell lines. Methods: We utilized a variety of approaches to examine the role of arginase II in hypoxic (1% O2, 5% CO2) cellular proliferation. Results: Arginase II mRNA and protein levels were significantly increased in osteosarcoma cells exposed to hypoxia for 48 hours. There were twice as many viable cells following 48 hours of hypoxia than following 48 hours of normoxia (21% O2, 5% CO2). The addition of difluoromethylornithine (DFMO), a putative arginase inhibitor, prevented hypoxia-induced proliferation. Transfection of small interfering RNAs (siRNA) targeting arginase II resulted in knockdown of arginase II protein levels and prevented hypoxia-induced cellular proliferation. Conclusions: These data support our hypothesis that hypoxia increases proliferation of osteosarcoma cells in an arginase II-dependent manner. We speculate that arginase II may represent a therapeutic target in osteosarcoma.

scholarly journals Role of HIF-1, Siah-1 and SKN-1 in Inducing Adiposity for Caenorhabditis elegans under Hypoxic Conditions

2020 ◽  
Vol 12 (1) ◽  
pp. 51-6
Author(s):  
I Gede Widhiantara ◽  
Anak Agung Ayu Putri Permatasari ◽  
I Wayan Rosiana ◽  
I Wayan Putu Sutirtayasa ◽  
Ferbian Milas Siswanto
Keyword(s):  
Body Length ◽  
Hypoxia Inducible Factors ◽  
Fat Accumulation ◽  
C Elegans ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Upstream Regulator ◽  
Morphometry Analysis ◽  
Seven In Absentia

BACKGROUND: Hypoxia has been shown to be able to induce adiposity. However, the mechanism and factors involved in this effect still remains unclear. Hence, we sought to investigate the role of oxygensensitive factors regarding hypoxia-induced adiposity in nematode Caenorhabditis elegans.METHODS: The C. elegans were grown on nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50 at 20°C. The ratio of width/body length was measured using the morphometry analysis. Fat accumulation was examined using Sudan Black methods. Protein levels of sterol binding protein (SBP)-1 were assessed by immunoblotting. Lifespan assay was performed at 20°C and was monitored every two days.RESULTS: The results showed that of all mutant used, only hif-1 mutant which did not experience an increase in the ratio of width/body length (p>0.05) and fat accumulation (p>0.05), indicating that hypoxia-inducible factors (HIF)-1 plays an important role in the pathogenesis of hypoxia-induced adiposity. Both siah-1 and skn-1 mutants experienced SBP-1 protein elevation (p<0.05), and increased fat-6 mRNA expression (p<0.05) which were not experienced by a hif-1 mutant (p>0.05) further supporting that HIF-1 acts as an upstream regulator fromSBP-1.CONCLUSION: In general, the results of this study provide evidences of the involvement of the transcription factor HIF-1 in inducing adiposity under the hypoxic conditions. However, we did not find the involvement of seven in absentia homolog-1 (Siah-1) and skinhead-1 (SKN-1).KEYWORDS: hypoxia, adiposity, fat, HIF-1, Siah-1, SKN-1, C. elegans

scholarly journals Evidence of Paraoxonases 1, 2, and 3 Expression in Human Ovarian Granulosa Cells

Antioxidants ◽  
2021 ◽  
Vol 10 (10) ◽  
pp. 1504
Author(s):  
Irantzu Pérez-Ruiz ◽  
José-Ignacio Ruiz-Sanz ◽  
María-Luisa Hérnandez ◽  
Rosaura Navarro ◽  
Marcos Ferrando ◽  
...  
Keyword(s):  
Granulosa Cells ◽  
Protein Detection ◽  
Oocyte Retrieval ◽  
Cell Cycles ◽  
Protein Levels ◽  
Ovarian Granulosa Cells ◽  
Ovarian Cells ◽  
Reproduction Process ◽  
Polymerase Chain

Increasing evidence suggests that the antioxidant paraoxonase proteins, PON1, PON2, and PON3, have a role in reproduction and may be synthesized by ovarian cells. The aim of this work was to investigate whether human ovarian granulosa cells (GC) express paraoxonases 1, 2, and 3 (PON1, PON2, and PON3) at both the transcriptional and protein levels. Cells were purified from follicle samples of women undergoing ovarian stimulation at oocyte retrieval. We analyzed mRNA by polymerase chain reaction using specific primers for the different variants and quantified the proteins by Western blot using commercially available human recombinant PON proteins as standards. The protein subcellular distribution was determined by immunofluorescence and confocal microscopy and the cell cycles by flow cytometry. Thymidine was used for cellular synchronization at G1/S. Human hepatoma HepG2 and immortalized granulosa COV434 cell lines were used to optimize methodologies. mRNAs from PON1, the two variants of PON2, and PON3 were detected in GC. The cells actively secreted PON1 and PON3, as evidenced by the protein detection in the incubation medium. PON1 and PON3 were mainly distributed in the cytoplasm and notably in the nucleus, while PON2 colocalized with mitochondria. Subcellular nucleo-cytoplasmic distribution of PON1 was associated with the cell cycle. This is the first evidence describing the presence of mRNAs and proteins of the three members of the PON family in human ovarian GC. This study provides the basis of further research to understand the role of these proteins in GC, which will contribute to a better understanding of the reproduction process.

scholarly journals Protective role of estrogen against excessive erythrocytosis in Monge’s disease

2021 ◽  
Vol 53 (1) ◽  
pp. 125-135
Author(s):  
Priti Azad ◽  
Francisco C. Villafuerte ◽  
Daniela Bermudez ◽  
Gargi Patel ◽  
Gabriel G. Haddad
Keyword(s):  
High Altitude ◽  
Messenger Rna ◽  
Target Genes ◽  
Protective Role ◽  
Mrna Levels ◽  
Protein Levels ◽  
Hypoxic Conditions ◽  
Andean Population

AbstractMonge’s disease (chronic mountain sickness (CMS)) is a maladaptive condition caused by chronic (years) exposure to high-altitude hypoxia. One of the defining features of CMS is excessive erythrocytosis with extremely high hematocrit levels. In the Andean population, CMS prevalence is vastly different between males and females, being rare in females. Furthermore, there is a sharp increase in CMS incidence in females after menopause. In this study, we assessed the role of sex hormones (testosterone, progesterone, and estrogen) in CMS and non-CMS cells using a well-characterized in vitro erythroid platform. While we found that there was a mild (nonsignificant) increase in RBC production with testosterone, we observed that estrogen, in physiologic concentrations, reduced sharply CD235a+ cells (glycophorin A; a marker of RBC), from 56% in the untreated CMS cells to 10% in the treated CMS cells, in a stage-specific and dose-responsive manner. At the molecular level, we determined that estrogen has a direct effect on GATA1, remarkably decreasing the messenger RNA (mRNA) and protein levels of GATA1 (p < 0.01) and its target genes (Alas2, BclxL, and Epor, p < 0.001). These changes result in a significant increase in apoptosis of erythroid cells. We also demonstrate that estrogen regulates erythropoiesis in CMS patients through estrogen beta signaling and that its inhibition can diminish the effects of estrogen by significantly increasing HIF1, VEGF, and GATA1 mRNA levels. Taken altogether, our results indicate that estrogen has a major impact on the regulation of erythropoiesis, particularly under chronic hypoxic conditions, and has the potential to treat blood diseases, such as high altitude severe erythrocytosis.

A Statistical Study of Process Variables to Optimize a High Speed Curtain Coater: Part I

TAPPI Journal ◽  
2009 ◽  
Vol 8 (1) ◽  
pp. 20-26 ◽  
Author(s):  
PEEYUSH TRIPATHI ◽  
MARGARET JOYCE ◽  
PAUL D. FLEMING ◽  
MASAHIRO SUGIHARA
Keyword(s):  
Boundary Layer ◽  
Experimental Design ◽  
High Speed ◽  
Statistical Study ◽  
Process Variables ◽  
High Speeds ◽  
The Stability ◽  
Air Removal ◽  
Removal System

Using an experimental design approach, researchers altered process parameters and material prop-erties to stabilize the curtain of a pilot curtain coater at high speeds. Part I of this paper identifies the four significant variables that influence curtain stability. The boundary layer air removal system was critical to the stability of the curtain and base sheet roughness was found to be very important. A shear thinning coating rheology and higher curtain heights improved the curtain stability at high speeds. The sizing of the base sheet affected coverage and cur-tain stability because of its effect on base sheet wettability. The role of surfactant was inconclusive. Part II of this paper will report on further optimization of curtain stability with these four variables using a D-optimal partial-facto-rial design.

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