Lack of expression of the mRNA encoding a major protein of the mouse vas deferens after neonatal exposure to oestrogens

1991 ◽  
Vol 7 (1) ◽  
pp. 63-69 ◽  
Author(s):  
E. Pailhoux ◽  
A. Martinez ◽  
Ch. Jean-Faucher ◽  
G. Veyssière ◽  
Cl. Jean
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Vas Deferens ◽  
Secretory Protein ◽  
Neonatal Exposure ◽  
Major Protein ◽  
Mouse Vas Deferens ◽  
Adult Mice

ABSTRACT We have previously characterized an androgen-inducible secretory protein from the mouse vas deferens (MVDP), and a cDNA to its mRNA has been obtained. This report describes altered MVDP gene expression after neonatal exposure to oestrogens. As shown by immunohistochemistry and Western blot analysis, MVDP was missing in the vas deferens from adult mice neonatally exposed to oestrogens. Northern blot analysis showed that the expression of MVDP mRNA was also suppressed. Exogenous testosterone was unable to stimulate MVDP production (either message or protein) in neonatally oestrogenized males. The results suggest that the alterations in gene expression in the oestrogen-exposed vas deferens reflect changes in the programme of differentiation of the organ itself.

scholarly journals IL-8 and MCP Gene Expression and Production by LPS-Stimulated Human Corneal Stromal Cells

2012 ◽  
Vol 2012 ◽  
pp. 1-4 ◽  
Author(s):  
Roni M. Shtein ◽  
Susan G. Elner ◽  
Zong-Mei Bian ◽  
Victor M. Elner
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Stromal Cells ◽  
Blot Analysis ◽  
Time Course ◽  
Northern Blot Analysis ◽  
Statistical Significance ◽  
Dependent Manner ◽  
Corneal Inflammation ◽  
Chemotactic Protein

Purpose. To determine time course of effect of lipopolysaccharide (LPS) on production of interleukin-8 (IL-8) and monocyte chemotactic protein (MCP) by cultured human corneal stromal cells.Methods. Human corneal stromal cells were harvested from donor corneal specimens, and fourth to sixth passaged cells were used. Cell cultures were stimulated with LPS for 2, 4, 8, and 24 hours. Northern blot analysis of IL-8 and MCP gene expression and ELISA for IL-8 and MCP secretion were performed. ELISA results were analyzed for statistical significance using two-tailed Student'st-test.Results. Northern blot analysis demonstrated significantly increased IL-8 and MCP gene expression after 4 and 8 hours of exposure to LPS. ELISA for secreted IL-8 and MCP demonstrated statistically significant increases (P<0.05) after corneal stromal cell stimulation with LPS.Conclusions. This paper suggests that human corneal stromal cells may participate in corneal inflammation by secreting potent leukocyte chemotactic and activating proteins in a time-dependent manner when exposed to LPS.

Gene expression analysis of the pro-oestrous-stage rat uterus reveals neuroligin 2 as a novel steroid-regulated gene

2004 ◽  
Vol 16 (8) ◽  
pp. 763 ◽  
Author(s):  
Han-Seung Kang ◽  
Chae-Kwan Lee ◽  
Ju-Ran Kim ◽  
Seong-Jin Yu ◽  
Sung-Goo Kang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Expression Profiles ◽  
Expression Patterns ◽  
Oestrous Cycle ◽  
Mrna Levels ◽  
Rat Uterus ◽  
Ovx Rats

In the present study, differential gene expression in the uteri of ovariectomised (OVX) and pro-oestrous rats (OVX v. pro-oestrus pair) was investigated using cDNA expression array analysis. Differential uterine gene expression in OVX rats and progesterone (P4)-injected OVX rats (OVX v. OVX + P4 pair) was also examined. The uterine gene expression profiles of these two sets of animals were also compared for the effects of P4 treatment. RNA samples were extracted from uterine tissues and reverse transcribed in the presence of [α32P]-dATP. Membrane sets of rat arrays were hybridised with cDNA probe sets. Northern blot analysis was used to validate the relative gene expression patterns obtained from the cDNA array. Of the 1176 cDNAs examined, 23 genes showed significant (>two-fold) changes in expression in the OVX v. pro-oestrus pair. Twenty of these genes were upregulated during pro-oestrus compared with their expression in the OVX rat uterus. In the OVX v. OVX + P4 pair, 22 genes showed significant (>two-fold) changes in gene expression. Twenty of these genes were upregulated in the OVX + P4 animals. The genes for nuclear factor I–XI, afadin, neuroligin 2, semaphorin Z, calpain 4, cyclase-associated protein homologue, thymosin β-4X and p8 were significantly upregulated in the uteri of the pro-oestrus and OVX + P4 rats of both experimental pairs compared with the OVX rat uteri. These genes appear to be under the control of P4. One of the most interesting findings of the present study is the unexpected and marked expression of the neuroligin 2 gene in the rat uterus. This gene is expressed at high levels in the central nervous system and acts as a nerve cell adhesion factor. According to Northern blot analysis, neuroligin 2 gene expression was higher during the pro-oestrus and metoestrus stages than during the oestrus and dioestrus stages of the oestrous cycle. In addition, neuroligin 2 mRNA levels were increased by both 17β-oestradiol (E2) and P4, although P4 administration upregulated gene expression to a greater extent than injection of E2. These results indicate that neuroligin 2 gene expression in the rat uterus is under the control of both E2 and P4, which are secreted periodically during the oestrous cycle.

scholarly journals The Branch Point Enzyme of the Mevalonate Pathway for Protein Prenylation Is Overexpressed in theob/ob Mouse and Induced by Adipogenesis

2000 ◽  
Vol 20 (6) ◽  
pp. 2158-2166 ◽  
Author(s):  
David Vicent ◽  
Eleftheria Maratos-Flier ◽  
C. Ronald Kahn
Keyword(s):  
Skeletal Muscle ◽  
Western Blot ◽  
Branch Point ◽  
Northern Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Northern Blot Analysis ◽  
Geranylgeranyl Diphosphate ◽  
Protein Prenylation ◽  
Ggpp Synthase

ABSTRACT We have recently reported that skeletal muscle of theob/ob mouse, an animal model of genetic obesity with extreme insulin resistance, exhibits alterations in the expression of multiple genes. Analysis and cloning of a full-length cDNA of one of the overexpressed mRNAs revealed a 300-amino-acid protein that could be identified as the mouse geranylgeranyl diphosphate synthase (GGPP synthase) based on its homology to proteins cloned from yeast and fungus. GGPP synthase catalyzes the synthesis of all-trans-geranylgeranyl diphosphate (GGPP), an isoprenoid used for protein isoprenylation in animal cells, and is a branch point enzyme in the mevalonic acid pathway. Three mRNAs for GGPP synthase of 4.3, 3.2, and 1.7 kb were detected in Northern blot analysis. Western blot analysis of tissue homogenates using specific antipeptide antibodies revealed a single band of 34.8 kDa. Expression level of this protein in different tissues correlated with expression of the 4.3- and 3.2-kb mRNAs. GGPP synthase mRNA expression was increased 5- to 20-fold in skeletal muscle, liver, and fat of ob/obmice by Northern blot analysis. Western blot analysis also showed a twofold overexpression of the protein in muscle and fat but not in liver, where the dominant isoform is encoded by the 1.7-kb mRNA. Differentiation of 3T3-L1 fibroblasts into adipocytes induced GGPP synthase expression more than 20-fold. Using the immunoprecipitated protein, we found that mammalian GGPP synthase synthesizes not only GGPP but also its metabolic precursor farnesyl diphosphate. Thus, the expression of GGPP synthase is regulated in multiple tissues in obesity and is induced during adipocyte differentiation. Altered regulation in the synthesis of isoprenoids for protein prenylation in obesity might be a factor determining the ability of the cells to respond to hormonal stimulation requiring both Ras-related small GTPases and trimeric G protein-coupled receptors.

Abstract WMP106: Reversal of Tonic Inhibition Contributes to Recovery of Synaptic Function After Transient Focal Cerebral Ischemia

Stroke ◽  
2016 ◽  
Vol 47 (suppl_1) ◽  
Author(s):  
James E Orfila ◽  
Robert M Dietz ◽  
Himmat Grewal ◽  
Takeru Shimizu ◽  
Frank F Strnad ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Gabaa Receptors ◽  
Western Blot Analysis ◽  
Hippocampal Slices ◽  
Long Term Potentiation ◽  
The United States ◽  
Synaptic Function ◽  
Gaba Transporter ◽  
Adult Mice

Introduction: Ischemic stroke is the fourth leading cause of death in the United States and is increasingly being recognized as a disease that occurs in people of all ages, not just the elderly. Studies suggest that the immature developing brain may have a greater degree of plasticity compared to the adult, thereby enhancing functional recovery to a greater extent during development. Hypothesis: Pediatric mice exhibit greater recovery from hippocampal synaptic function following experimental stroke than adults. Methods: Extracellular field recordings of CA1 neurons were performed in acute hippocampal slices prepared at, 7 or 30 days after recovery from middle cerebral artery occlusion (MCAO). A behavioral fear conditioning paradigm was done to evaluate contextual memory in both pediatric and adult mice 30 days after MCAO. α5 GABAA receptors and GABA transporter expression was evaluated by western blot analysis. Results: In adult mice following MCAO, hippocampal long-term potentiation (LTP), defined as an increase in synaptic strength, remained impaired for at least 30 days in the ipsilateral and contralateral, non-injured hemisphere. However, in pediatric mice following MCAO, LTP was only impaired in the ipsilateral side 7 days after MCAO and showed full recovery of synaptic function at 30 days. Behavioral data confirmed these data, showing that only adult mice displayed memory deficits 30 days after MCAO. L655,708 (100nM), an inverse agonist selective for α5 GABAA receptors, rescued LTP in acute slices in both pediatric and adult mice at all-time points tested. Western blot analysis failed to identify any changes in α5 GABAA receptors or GABA transporter (GAT1 or GAT3) levels after MCAO. Conclusion: The present study demonstrates that transient focal ischemia causes functional impairment in the hippocampus and that recovery of behavioral and synaptic function is more robust in the young brain. In addition, inhibition of tonic GABA activity rescues synaptic function, indicating that targeting of excessive GABA activity may provide a therapeutic approach to improve cognitive recovery after stroke.

Gender and transcriptional regulation of NO synthase and ET-1 in porcine aortic endothelial cells

1997 ◽  
Vol 273 (4) ◽  
pp. H1962-H1967 ◽  
Author(s):  
Xiaofang Wang ◽  
Dustan A. Barber ◽  
Debra A. Lewis ◽  
Christopher G. A. McGregor ◽  
Gary C. Sieck ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Endothelial Cells ◽  
Western Blot ◽  
Northern Blot ◽  
Blot Analysis ◽  
Northern Blot Analysis ◽  
Ovarian Hormones ◽  
Intact Male ◽  
Male And Female ◽  
Aortic Endothelial Cells

Experiments were designed to determine whether normal fluctuations in sex steroid hormones alter gene transcription for endothelial nitric oxide synthase (NOS) and preproendothelin-1 (prepro-ET-1). Aortic endothelial cells were removed from adult, gonadally intact male and female or ovariectomized Yorkshire pigs. Endothelial cells were prepared for Northern blot analysis, Western blot analysis or enzyme activity. Nitric oxide products (NOx) and endothelin-1 (ET-1) in plasma were measured by chemiluminescence and radioimmunoassay, respectively. Northern blot analysis identified single bands corresponding to endothelial NOS and prepro-ET-1. Quantification of the blots showed an increase in expression of mRNA for both endothelial NOS and prepro-ET-1 in ovariectomized pigs compared with gonadally intact male and female pigs. There were no differences in amount of endothelial NOS protein identified by Western blot analysis among groups. On the contrary, plasma concentrations of NOx were significantly decreased in ovariectomized pigs, and there were no differences either in the concentrations of ET-1 in the plasma or extracts from the coronary arteries. These results suggest that expression of endothelial NOS and prepro-ET-1 may be regulated at transcriptional level by ovarian hormones. In addition, the ovarian hormones may regulate production of these endothelium-derived factors at the posttranscriptional level.

Growth Factor Independence 1 (Gfi-1) Plays a Role in Mediating Specific Granule Deficiency (SGD) in a Patient Lacking an Abnormality in the C/EBPε Gene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3546-3546
Author(s):  
Arati Khanna-Gupta ◽  
Terry Zibello ◽  
Hong Sun ◽  
Laurence A. Boxer ◽  
Nancy Berliner
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Transcription Factor ◽  
Growth Factor ◽  
Western Blot ◽  
Blot Analysis ◽  
Germline Mutations ◽  
Coding Region ◽  
Blood Neutrophils ◽  
Specific Granule

Abstract Neutrophil specific granule deficiency (SGD) is a rare congenital disorder marked by recurrent bacterial infections of the skin and respiratory system. Neutrophils from SGD patients lack secondary and tertiary granules and their content proteins and exhibit defects in chemotaxis and bactericidal activity. A mouse model deficient for the transcription factor CCAAT/enhancer binding protein epsilon (C/EBPε) manifests a similar phenotype as SGD patients and prompted examination of the human C/EBPε gene for mutation in this disease. Mutations in the C/EBPε gene have been identified by others in two patients with SGD, resulting in loss of gene expression. However, other patients with a similar disease phenotype have a normal C/EBPε coding sequence, suggesting that other genetic abnormalities in myelopoiesis can lead to SGD. Studies in our laboratory on one such patient lacking a C/EBPε mutation demonstrated an elevated level of the C/EBPε protein in the patient’s peripheral blood neutrophils as compared to the level in normal control neutrophils. Microarray analysis of this patient’s bone marrow compared with that of a normal control revealed, among other genes, elevated levels of the transcription factors C/EBPε and PU.1. As a consequence, several PU.1 target genes showed increased expression in the SGD patient bone marrow. This observation was confirmed by both real time PCR and western blot analysis. PU.1 is a hematopoietic-specific transcription factor belonging to the Ets family of DNA binding proteins and plays a critical role in B-cell, macrophage and late stage neutrophil development. Sequence analysis of the PU.1 gene from our SGD patient however, did not reveal any mutation in the coding region of the gene. Western blot analysis of nuclear extracts prepared from peripheral blood neutrophils from this patient did however reveal significantly decreased levels of the transcription factor Gfi-1 (Growth factor independent-1), although no mutation has been found thus far in the coding region of the Gfi-1 gene from the SGD patient. Gfi-1 belongs to a family of zinc finger containing transcriptional repressor oncoproteins. Mice lacking Gfi-1 were found not only to be neutropenic, but also demonstrated defects in neutrophil differentiation, including loss of neutrophil secondary and tertiary granule proteins, reminiscent of SGD. More recently, heterozygous germline mutations of Gfi-1 were shown to cause severe congenital neutropenia (SCN) in humans. It has been suggested that Gfi1 represses neutrophil elastase (Ela2), germline mutations within which are a major contributor to hereditary neutropenias. Our data suggest that decreased levels of Gfi1 in our SGD patient result in increased levels of PU.1 and C/EBPε; an effect consistent with observations first made in the neutrophils of Gfi-1-null mice. The increased PU.1 levels might then act to sequester C/EBPε protein via direct protein-protein interaction. This in turn could explain the loss of secondary granule protein gene expression in the SGD patient by inducing functional C/EBPε deficiency.

scholarly journals Endoplasmic Reticulum Stress is Involved in DFMO Attenuating Isoproterenol-Induced Cardiac Hypertrophy in Rats

2016 ◽  
Vol 38 (4) ◽  
pp. 1553-1562 ◽  
Author(s):  
Yan Lin ◽  
Xiaojie Zhang ◽  
Wei Xiao ◽  
Bo Li ◽  
Jun Wang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Endoplasmic Reticulum ◽  
Endoplasmic Reticulum Stress ◽  
Cardiac Hypertrophy ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cardiac Fibrosis ◽  
Regulation Of Gene Expression ◽  
Tunel Staining

Background/Aims: Studies performed in experimental animals have shown that polyamines contribute to several physiological and pathological processes, including cardiac hypertrophy. This involves an increase in ornithine decarboxylase (ODC) activity and intracellular polyamines associated with regulation of gene expression. Difluoromethylornithine (DFMO), an irreversible inhibitor of ODC, has attracted considerable interest for its antiproliferative role, which it exerts through inhibition of the polyamine pathway and cell turnover. Whether DFMO attenuates cardiac hypertrophy through endoplasmic reticulum stress (ERS) is unclear. Methods: Myocardial hypertrophy was simulated by isoproterenol (ISO). Polyamine depletion was achieved using DFMO. Hypertrophy was estimated using the heart/body index and atrial natriuretic peptide (ANP) gene expression. Cardiac fibrosis and apoptosis were measured by Masson and TUNEL staining. Expression of ODC and spermidine/spermine N1-acetyltransferase (SSAT) were analyzed via real-time PCR and Western blot analysis. Protein expression of ERS and apoptosis factors were analyzed using Western blot analysis. Results: DFMO treatments significantly attenuated hypertrophy and apoptosis induced by ISO in cardiomyocytes. DFMO down-regulated the expression of ODC, glucose-regulated protein 78 (GRP78), C/EBP homologous protein (CHOP), cleaved caspase-12, and Bax and up-regulated the expression of SSAT and Bcl-2. Finally, these changes were partially reversed by the addition of exogenous putrescine. Conclusion: The data presented here suggest that polyamine depletion could inhibit cardiac hypertrophy and apoptosis, which is closely related to the ERS pathway.

Secretoneurin is a potential paracrine factor from lactotrophs stimulating gonadotropin release in the goldfish pituitary

2010 ◽  
Vol 299 (5) ◽  
pp. R1290-R1297 ◽  
Author(s):  
E. Zhao ◽  
Caleb L. Grey ◽  
Dapeng Zhang ◽  
Jan A. Mennigen ◽  
Ajoy Basak ◽  
...  
Keyword(s):  
Gene Expression ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Time Course ◽  
Pcr Analysis ◽  
Pituitary Cells ◽  
Mrna Levels ◽  
Paracrine Factor ◽  
Lh Release

Secretoneurin (SN) is a functional neuropeptide derived from the evolutionarily conserved part of precursor protein secretogranin II (SgII). In the time course study, SN (10 nM) stimulates luteinizing hormone (LH) production and secretion after 6 h of static incubation of goldfish pituitary cells. Due to the existence of SN-immunoreactivity (SN-IR) in goldfish lactotrophs, endogenous SN might exert a paracrine effect on LH in the pituitary. In an in vitro immunoneutralization experiment, coincubation with anti-SN antiserum reduces the stimulatory effect of salmon gonadotropin-releasing hormone (sGnRH) on LH release by 64%. Using Western blot analysis, we demonstrate that sGnRH significantly increases the expression of the major SgII-derived peptide (∼57 kDa, with SN-IR) and prolactin (PRL) after 12 h in the static culture of goldfish pituitary cells. Furthermore, there exists a significant correlation between the levels of these two proteins ( R = 0.76, P = 0.004). Another ∼30 kDa SgII-derived peptide containing SN is only observed in sGnRH-treated pituitary cells. Consistent with the Western blot analysis results, real-time RT-PCR analysis shows that a 12-h treatment with sGnRH induced 1.6- and 1.7-fold increments in SgII and PRL mRNA levels, respectively. SgII gene expression was also associated with PRL gene expression ( R = 0.66; P = 0.02). PRL cells loaded with the calcium-sensitive dye, fura 2/AM, respond to sGnRH treatment with increases in intracellular Ca2+ concentration level, suggesting a potential mechanism of GnRH on PRL cells and thus SgII processing and SN secretion. Taken together, endogenous lactotroph-generated SN, under the control of hypothalamic GnRH, exerts a paracrine action on neighboring gonadotrophs to stimulate LH release.

Androgen regulation of the mRNA encoding a major protein of the mouse vas deferens

1990 ◽  
Vol 72 (3) ◽  
pp. 201-211 ◽  
Author(s):  
A. Martinez ◽  
E. Pailhoux ◽  
M. Berger ◽  
Cl. Jean
Keyword(s):  
Vas Deferens ◽  
Major Protein ◽  
Mouse Vas Deferens ◽  
Androgen Regulation

Myc-epitope tagged proteins detected with the 9E10 antibody in immunofluorescence and immunoprecipitation assays but not in Western blot analysis

1998 ◽  
Vol 76 (1) ◽  
pp. 125-128 ◽  
Author(s):  
Huizhou Fan ◽  
Cristy Villegas ◽  
Arthur K Chan ◽  
Jim A Wright
Keyword(s):  
Gene Expression ◽  
Monoclonal Antibody ◽  
Recombinant Protein ◽  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Immunofluorescence Assay ◽  
Expression Vectors ◽  
Epitope Recognition ◽  
In Cells

A human Myc epitope is frequently used to tag proteins for expression experiments in nonhuman cells. We used the monoclonal 9E10 antibody specific for this epitope to analyse the expression of four proteins carrying the Myc tag in cells transfected with expression vectors. While all four proteins can be detected by immunofluorescence and immunoprecipitation assays, surprisingly, only two proteins could be detected in Western blot analysis, indicating that epitope recognition by the monoclonal antibody can be blocked in some membrane-retained ectopic proteins. Other techniques such as immunofluorescence and immunoprecipitation assays can be successfully used with the 9E10 antibody to determine potential expression of Myc-tagged proteins.Key words: recombinant protein, Myc epitope, 9E10, Western blot, gene expression, immunofluorescence assay, immunoprecipitation.

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