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Sample records for receptor expression level

  1. Endothelin A and B receptors change their expression levels during development of human placental villi.

    PubMed

    Cervar, M; Huppertz, B; Barth, S; Hahn, T; Weiss, U; Kaufmann, P; Desoye, G

    2000-01-01

    Endothelin receptors have recently been found in non-vascular tissues including the human placenta. The present study investigated developmental changes in location and expression levels of endothelin A and B receptors (ETA-R, ETB-R) in human placentae and isolated trophoblast by comparing first and third trimester tissues. In the first trimester all cells and tissues were immunolabelled for ETA-R and ETB-R, whereas in third trimester placentae the syncytiotrophoblast (ETA-R, ETB-R) and macrophages (ETA-R) were unstained. Immunoblotting for both receptors revealed up to three bands at 33-35, 50 and 75 kDa, respectively, which were differentially present in the first and third trimester. Pre-adsorption of antibodies with oligopeptides used for antigen-generation weakened the immunoreactions. ETA-R protein levels decreased (P< 0.05) in total villous tissue and isolated trophoblast, whereas ETB-R was unchanged. ETB-R transcripts (RT-PCR) prevailed in both stages and tissues, but in contrast to the protein levels its preponderance decreased from first trimester to term in villous tissue (P< 0.01), because of a four to five-fold increase in ETA-R and only a two-fold (P< 0.05) increase in ETB-R mRNA levels (P< 0.01). We conclude that ET receptor location, intracellular processing and expression levels in human villous tissue change between the first and third trimester. This may reflect changing functions of ET-1 during placental development. PMID:10940204

  2. Correlated expression levels of endothelin receptor B and Plexin C1 in melanoma

    PubMed Central

    Kumasaka, Mayuko Y; Yajima, Ichiro; Iida, Machiko; Takahashi, Hiro; Inoue, Yuji; Fukushima, Satoshi; Ihn, Hironobu; Takeda, Kozue; Naito, Yuji; Yoshikawa, Toshikazu; Kato, Masashi

    2015-01-01

    Discussion concerning the effect of endothelin receptor B (Ednrb) on melanoma continues because Ednrb has been reported to have both tumor promoting and suppressive effects for melanoma. In order to examine Ednrb-related signaling in melanomagenesis, DNA microarray analysis for a melanoma from a RFP/RET-transgenic mouse (RET-mouse) and a melanoma from an Ednrb-heterozygously deleted RET-mouse [Ednrb(+/-);RET-mouse], in both of which melanoma spontaneously develops, was performed in this study. We found that the expression level of Plexin C1 (PlxnC1), a suppressor for melanoma, in a melanoma from an Ednrb(+/-);RET-mouse was drastically decreased compared to that in a melanoma from a RET-mouse. Therefore, we further examined the correlation between Ednrb and PlxnC1 expression levels in melanomas. PlxnC1 transcript expression levels in melanomas from Ednrb(+/-);RET-mice were lower than those in melanomas from RET-mice. A strong correlation between Ednrb and PlxnC1 transcript expression levels (R = 0.78, p < 0.01) was also found in melanomas from both RET-mice and Ednrb(+/-);RET-mice. Correspondingly, there was a significant correlation between transcript (R = 0.80; p < 0.01) and protein (R = 0.60; p < 0.01) expression levels of EDNRB and PLXNC1 in human primary melanomas. Together with our results showing that the expression level of PLXNC1 transcript was reduced in EDNRB-depleted human melanoma cells, our results showing positively correlated expression levels of Ednrb/EDNRB and PlxnC1/PLXNC1 in melanoma suggest that PlxnC1/PLXNC1 is involved in the Ednrb/EDNRB-mediated suppressive effect on melanoma. PMID:26045990

  3. [Expression of erythropoietin receptor in leukemia cells and relation of erythropoietin level with leukemic anemia].

    PubMed

    Feng, Mei; Li, Yu-Cui

    2008-12-01

    This study was purposed to investigate the expression of erythropoietin receptor (EPOR) in leukemic cells and the relationship of serum erythropoietin level with anemia in acute leukemia patients, so as to provide a new theoretical basis for the cytokine therapy in acute leukemia with anemia. The EPOR in 30 AL patients was detected by using reverse transcription polymerase chain reaction (RT-PCR), the level of serum erythropoietin was detected by chemiluminescence analysis, the hemoglobin level was assayed by automatic blood counting instrument. The results indicated that EPOR was expressed in 18 out of 30 AL patients, the expression rate of EPOR in AL patients was 60%, however, but the EPOR expression rate in AML was 61.9% (13/21) and 55.6% (5/9) in ALL, the EPOR expression rate was no significant difference between AML and ALL. The EPOR expression rate was significantly lower than that in control group (86.7%) (p<0.05). The relative level of EPOR expression in AML was higher than that in ALL (p<0.05), the average level of EPOR expression in AL was significantly lower than that in control group (p<0.01). The level of sEPO in 30 AL patients was significantly higher than that in control group (p<0.01), and there was negative correlation between the levels of sEPO and Hb (p<0.01). It is concluded that the EPOR is expressed in cells of AL, but the expressive level is low. The EPOR expression rate shows no significant difference between AML and ALL. The mechanism of negative feedback to anemia in acute leukemia is intact. Anemia of acute leukemia is not completely associated with inadequate erythropoietin production and relates to hemopoiesis defect that considered as the main reason. Recombinant human erythropoietin is widely used in treatment of anemia caused by acute leukemia. Whether the treatment with rh-EPO for acute leukemia with anemia will enhance the proliferation of leukemia cells, this problem should be explored further. PMID:19099624

  4. Androgen Receptors in a Cichlid Fish, Astatotilapia burtoni: Structure, Localization, and Expression Levels

    PubMed Central

    HARBOTT, LENE K.; BURMEISTER, SABRINA S.; WHITE, RICHARD B.; VAGELL, MIKE; FERNALD, RUSSELL D.

    2009-01-01

    Androgens are an important output of the hypothalamic-pituitary-gonadal (HPG) axis that controls reproduction in all vertebrates. In male teleosts two androgens, testosterone and 11-ketotestosterone, control sexual differentiation and development in juveniles and reproductive behavior in adults. Androgenic signals provide feedback at many levels of the HPG axis, including the hypothalamic neurons that synthesize and release gonadotropin-releasing hormone 1 (GnRH1), but the precise cellular site of androgen action in the brain is not known. Here we describe two androgen receptor subtypes, ARα and ARβ, in the cichlid Astatotilapia burtoni and show that these subtypes are differentially located throughout the adult brain in nuclei known to function in the control of reproduction. ARα was expressed in the ventral part of the ventral telencephalon, the preoptic area (POA) of the hypothalamus and the ventral hypothalamus, whereas ARβ was more widely expressed in the dorsal and ventral telencephalon, the POA, and the ventral and dorsal hypothalamus. We provide the first evidence in any vertebrate that the GnRH1-releasing neurons, which serve as the central control point of the HPG axis, express both subtypes of AR. Using quantitative real-time PCR, we show that A. burtoni AR subtypes have different expression levels in adult tissue, with ARα showing significantly higher expression than ARβ in the pituitary, and ARβ expressed at a higher level than ARα in the anterior and middle brain. These data provide important insight into the role of androgens in regulating the vertebrate reproductive axis. PMID:17614300

  5. TREM-2 Receptor Expression Increases with 25(OH)D Vitamin Serum Levels in Patients with Pulmonary Sarcoidosis

    PubMed Central

    Bucova, Maria; Suchankova, Magda; Tibenska, Elena; Tedlova, Eva; Demian, Juraj; Majer, Ivan; Novosadova, Helena; Tedla, Miroslav

    2015-01-01

    TREM-1 and TREM-2 molecules are members of the TREM transmembrane glycoproteins. In our previous study we identified increased expressions of TREM-1 and TREM-2 receptors in pulmonary sarcoidosis (PS). Only a few studies concerning the association between vitamin D and TREM receptor expression can be found. The aim of our current study was to determine the association between the levels of an inactive form of 25(OH)D vitamin and TREM-1 and TREM-2 receptor expressions. We have detected low levels of 25(OH)D vitamin in 79% of PS patients. Only 21% of patients had normal serum level of 25(OH)D vitamin with values clustered within the low-normal range. The most striking findings were the increased TREM-2 expressions on myeloid cells surfaces in BALF of PS patients with normal 25(OH)D vitamin serum levels compared with those with its decreased levels. The total number of TREM-2 positive cells was 5.7 times higher and the percentage of TREM-2 positive cells was also significantly increased in BALF of PS patients with normal compared to PS patients with low 25(OH)D vitamin serum levels. A significant correlation between total TREM-2 expression and vitamin D levels has been detected too. However, we have not detected similar differences in TREM-1expression and 25(OH)D vitamin serum levels. PMID:26166951

  6. TREM-2 Receptor Expression Increases with 25(OH)D Vitamin Serum Levels in Patients with Pulmonary Sarcoidosis.

    PubMed

    Bucova, Maria; Suchankova, Magda; Tibenska, Elena; Tedlova, Eva; Demian, Juraj; Majer, Ivan; Novosadova, Helena; Tedla, Miroslav

    2015-01-01

    TREM-1 and TREM-2 molecules are members of the TREM transmembrane glycoproteins. In our previous study we identified increased expressions of TREM-1 and TREM-2 receptors in pulmonary sarcoidosis (PS). Only a few studies concerning the association between vitamin D and TREM receptor expression can be found. The aim of our current study was to determine the association between the levels of an inactive form of 25(OH)D vitamin and TREM-1 and TREM-2 receptor expressions. We have detected low levels of 25(OH)D vitamin in 79% of PS patients. Only 21% of patients had normal serum level of 25(OH)D vitamin with values clustered within the low-normal range. The most striking findings were the increased TREM-2 expressions on myeloid cells surfaces in BALF of PS patients with normal 25(OH)D vitamin serum levels compared with those with its decreased levels. The total number of TREM-2 positive cells was 5.7 times higher and the percentage of TREM-2 positive cells was also significantly increased in BALF of PS patients with normal compared to PS patients with low 25(OH)D vitamin serum levels. A significant correlation between total TREM-2 expression and vitamin D levels has been detected too. However, we have not detected similar differences in TREM-1expression and 25(OH)D vitamin serum levels. PMID:26166951

  7. Reduction of EGF receptor levels in human tumor cells transfected with an antisense RNA expression vector

    SciTech Connect

    Yamada, Hirotomo; Koizumi, Shinji; Kimura, Masami ); Shimizu, Nobuyoshi )

    1989-09-01

    An expression vector was constructed from part of pSV2neo with the 3{prime}-ClaI fragment of the epidermal growth factor (EGF) receptor cDNA inserted in an inverted orientation downstream from the human metallothionein (MT) IIa promoter. The human squamous carcinoma cell line NA, which overproduces EGF receptor, was transfected with this vector and selected for resistance to the neomycin derivative G418. One of the stable transfectants had a 90% reduction cell-surface EGF receptor in response to ZnSO{sub 4}. The nascent EGF receptor peptide was also decreased with concurrent induction of MT mRNA. These data suggest that the antisense transcript regulated by the MT promoter inhibits the expression of the endogenous EGF receptor genes. Although no transcripts from the antisense gene were detected, the results indicate that transfection with the antisense vector provides a technique by which to modulate the number of EGF receptors on the cell surface of squamous cell carcinomas.

  8. Polymorphisms in the Tumor Necrosis Factor Receptor Genes Affect the Expression Levels of Membrane-Bound Type I and Type II Receptors

    PubMed Central

    Sennikov, Sergey V.; Vasilyev, Filipp F.; Lopatnikova, Julia A.; Shkaruba, Nadezhda S.; Silkov, Alexander N.

    2014-01-01

    The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNFα receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP −609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP −1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14+ monocytes compared to individuals with the GC genotype. The frequency differences in the CD3+ and CD19+ cells expressing TNFRII in relation to SNP −1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP −3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14+ cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI −609GT + TNFRII −3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNFα receptors and TNFα-mediated signaling. PMID:24782596

  9. Short-term sleep deprivation impairs spatial working memory and modulates expression levels of ionotropic glutamate receptor subunits in hippocampus.

    PubMed

    Xie, Meilan; Yan, Jie; He, Chao; Yang, Li; Tan, Gang; Li, Chao; Hu, Zhian; Wang, Jiali

    2015-06-01

    Hippocampus-dependent learning memory is sensitive to sleep deprivation (SD). Although the ionotropic glutamate receptors play a vital role in synaptic plasticity and learning and memory, however, whether the expression of these receptor subunits is modulated by sleep loss remains unclear. In the present study, western blotting was performed by probing with specific antibodies against the ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunits GluA1, GluA2, GluA3, and against the N-methyl-d-aspartate (NMDA) glutamate receptor subunits GluN1, GluN2A, GluN2B. In hippocampus, down regulation of surface GluA1 and GluN2A surface expression were observed in both SD groups. However, surface expression level of GluA2, GluA3, GluN1 and GluN2B was significantly up-regulated in 8h-SD rats when compared to the 4h-SD rats. In parallel with the complex changes in AMPA and NMDA receptor subunit expressions, we found the 8h-SD impaired rat spatial working memory in 30-s-delay T-maze task, whereas no impairment of spatial learning was observed in 4h-SD rats. These results indicate that sleep loss alters the relative expression levels of the AMPA and NMDA receptors, thus affects the synaptic strength and capacity for plasticity and partially contributes to spatial memory impairment. PMID:25732956

  10. Evaluation of discoidin domain receptor-2 (DDR2) expression level in normal, benign, and malignant human prostate tissues

    PubMed Central

    Azemikhah, Mitra; Ashtiani, Hamidreza Ahmadi; Aghaei, Mahmoud; Rastegar, Hosein

    2015-01-01

    Discoidin domain receptor (DDR) is a new member of the receptor tyrosine kinase family. There are two isoforms of discoidin domain receptor (DDR), DDR1 and DDR2. These receptors play a major role in the adhesion, motility and cell proliferation. Due to the important role of DDR2 in the development of tumor extension, this receptor is pivotal in the field of carcinogenesis. The aim of this study was to investigate the mRNA and protein expression of DDR2, in the malignant, benign prostatic hyperplasia (BPH) and normal tissues of patients with prostate cancer. In this study the gene and protein expression of DDR2 in adjacent normal (n=40), BPH (n=40), and malignant (n=40) prostate tissue were measured using real-time PCR and Western blotting. Then, the correlation of DDR2 gene and protein expression with prognostic factors such as age, tumor grade, tumor stage, lymph node involvement, and serum prostate-specific antigen (PSA) concentration were evaluated. The relative mRNA and protein expression level of DDR2 in malignant and benign prostate tissue was significantly higher than those of adjacent normal tissues (P<0.01). This expression was found to increase approximately 3.5 and 2.1 fold for mRNA and protein levels, respectively. Spearman test indicated a significant correlation between DDR2 mRNA and protein expression with prognostic factors such as tumor grade, stage, lymph node involvement, and serum PSA concentration. However, significant correlation with age was not observed. These findings suggest that DDR2 is a cancer-related gene associated with the aggressive progression of prostate cancer patients. PMID:26600862

  11. Evaluation of discoidin domain receptor-2 (DDR2) expression level in normal, benign, and malignant human prostate tissues.

    PubMed

    Azemikhah, Mitra; Ashtiani, Hamidreza Ahmadi; Aghaei, Mahmoud; Rastegar, Hosein

    2015-01-01

    Discoidin domain receptor (DDR) is a new member of the receptor tyrosine kinase family. There are two isoforms of discoidin domain receptor (DDR), DDR1 and DDR2. These receptors play a major role in the adhesion, motility and cell proliferation. Due to the important role of DDR2 in the development of tumor extension, this receptor is pivotal in the field of carcinogenesis. The aim of this study was to investigate the mRNA and protein expression of DDR2, in the malignant, benign prostatic hyperplasia (BPH) and normal tissues of patients with prostate cancer. In this study the gene and protein expression of DDR2 in adjacent normal (n=40), BPH (n=40), and malignant (n=40) prostate tissue were measured using real-time PCR and Western blotting. Then, the correlation of DDR2 gene and protein expression with prognostic factors such as age, tumor grade, tumor stage, lymph node involvement, and serum prostate-specific antigen (PSA) concentration were evaluated. The relative mRNA and protein expression level of DDR2 in malignant and benign prostate tissue was significantly higher than those of adjacent normal tissues (P<0.01). This expression was found to increase approximately 3.5 and 2.1 fold for mRNA and protein levels, respectively. Spearman test indicated a significant correlation between DDR2 mRNA and protein expression with prognostic factors such as tumor grade, stage, lymph node involvement, and serum PSA concentration. However, significant correlation with age was not observed. These findings suggest that DDR2 is a cancer-related gene associated with the aggressive progression of prostate cancer patients. PMID:26600862

  12. Olfactory discrimination varies in mice with different levels of α7-nicotinic acetylcholine receptor expression

    PubMed Central

    Hellier, Jennifer L.; Arevalo, Nicole L.; Blatner, Megan J.; Dang, An K.; Clevenger, Amy C.; Adams, Catherine E.; Restrepo, Diego

    2010-01-01

    Previous studies have shown that schizophrenics have decreased expression of α7-nicotinic acetylcholine (α7) receptors in the hippocampus and other brain regions, paranoid delusions, disorganized speech, deficits in auditory gating (i.e., inability to inhibit neuronal responses to repetitive auditory stimuli), and difficulties in odor discrimination and detection. Here we use mice with decreased α7 expression that also show a deficit in auditory gating to determine if these mice have similar deficits in olfaction. In the adult mouse olfactory bulb (OB), α7 expression localizes in the glomerular layer; however, the functional role of α7 is unknown. We show that inbred mouse strains (i.e., C3H and C57) with varying α7 expression (e.g., α7 wild-type [α7+/+], α7 heterozygous knock-out [α7+/−] and α7 homozygous knockout mice [α7−/−]) significantly differ in odor discrimination and detection of chemically related odorant pairs. Using [125I] α-bungarotoxin (α-BGT) autoradiography, α7 expression was measured in the OB. As previously demonstrated, α-BGT binding was localized to the glomerular layer. Significantly more expression of α7 was observed in C57 α7+/+ mice compared to C3H α7+/+ mice. Furthermore, C57 α7+/+ mice were able to detect a significantly lower concentration of an odor in a mixture compared to C3H α7+/+ mice. Both C57 and C3H α7+/+ mice discriminated between chemically related odorants sooner than α7+/− or α7−/− mice. These data suggest that α7-nicotinic-receptors contribute strongly to olfactory discrimination and detection in mice and may be one of the mechanisms producing olfactory dysfunction in schizophrenics. PMID:20713028

  13. Low level expression of glycine receptor beta subunit transgene is sufficient for phenotype correction in spastic mice.

    PubMed Central

    Hartenstein, B; Schenkel, J; Kuhse, J; Besenbeck, B; Kling, C; Becker, C M; Betz, H; Weiher, H

    1996-01-01

    Mutations in inhibitory glycine receptor (GlyR) subunit genes are associated with neuromotor diseases in man and mouse. To use the potential of the mouse mutants as animal models of human disease, we altered GlyR levels in mutant mice and studied their phenotype. A transgene coding for the beta subunit of the rat GlyR was introduced into the genetic background of the spa mutation, which is characterized by low endogenous expression levels of the beta subunit and a dramatic neuromotor phenotype. The resulting transgenic mice expressed the beta subunit mRNA at intermediate levels, and their phenotype was rescued. This provides formal proof for the casual relationship between GlyR beta gene mutation and motor disease, and indicates that a low level of beta gene expression (25% of normal) is sufficient for proper functioning of glycinergic synapses. Images PMID:8635460

  14. Leptin receptor gene expression and number in the brain are regulated by leptin level and nutritional status.

    PubMed

    Mitchell, Sharon E; Nogueiras, Ruben; Morris, Amanda; Tovar, Sulay; Grant, Christine; Cruickshank, Morven; Rayner, D Vernon; Dieguez, Carlos; Williams, Lynda M

    2009-07-15

    Hormone potency depends on receptor availability, regulated via gene expression and receptor trafficking. To ascertain how central leptin receptors are regulated, the effects of leptin challenge, high-fat diet, fasting and refeeding were measured on leptin receptor number and gene expression. These were measured using quantitative (125)I-labelled leptin in vitro autoradiography and in situ hybridisation, respectively. Ob-R (all forms of leptin receptor) expression in the choroid plexus (CP) was unchanged by high-fat diet or leptin challenge, whereas fasting increased but refeeding failed to decrease expression. (125)I-labelled leptin binding to the CP was increased by fasting and returned to basal levels on refeeding. (125)I-Labelled leptin was reduced by leptin challenge and increased by high-fat feeding. Ob-Rb (signalling form) in the arcuate (ARC) and ventromedial (VMH) nuclei was increased after fasting and decreased by refeeding. Leptin challenge increased Ob-Rb expression in the ARC, but not after high-fat feeding. In general, changes in gene expression in the ARC and VMH appeared to be largely due to changes in area rather than density of labelling, indicating that the number of cells expressing Ob-Rb was the parameter that contributed most to these changes. Leptin stimulation of suppressor of cytokine signalling 3 (SOCS3), a marker of stimulation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway, was unchanged after high-fat diet. Thus, early loss of leptin sensitivity after high-fat feeding is unrelated to down-regulation of leptin receptor expression or number and does not involve the JAK/STAT pathway. The effect of leptin to decrease (125)I-labelled leptin binding and the loss of ability of leptin to up-regulate Ob-Rb expression in the ARC after high-fat feeding offer potential mechanisms for the development of leptin insensitivity in response to both hyperleptinaemia and high-fat diet. PMID:19491239

  15. Do fasudil and Y-27632 affect the level of transient receptor potential (TRP) gene expressions in breast cancer cell lines?

    PubMed

    Gogebakan, Bulent; Bayraktar, Recep; Suner, Ali; Balakan, Ozan; Ulasli, Mustafa; Izmirli, Muzeyyen; Oztuzcu, Serdar; Camci, Celaletdin

    2014-08-01

    Breast cancer (BC) is the most frequent cancer type in women, and the mortality rate is high especially in metastatic disease. Ion channels such as the transient receptor potential (TRP) channels correlate with malignant growth and cancer progression. Hence, some authors have suggested that the expression levels of TRP channels may be used as a marker in the diagnosis and predicting the prognosis of BC. Also, in some recent studies, targeting TRP channels are suggested as a novel treatment strategy in BC. The aim of this study was to investigate the effect of two Rho-kinase (ROCK) inhibitors, fasudil and Y-27632, on the expression levels of TRP channel genes in breast cancer cell lines (ZR-75-1, MCF7, and MDA-MB-231) and breast epithelial cell line (hTERT-HME1). The expression levels of TRP genes were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). We found that fasudil had reduced the TRPC1, TRPV2 expression levels in the ZR-75-1, MCF7, and MDA-MB-231 cell lines. On the other hand, fasudil and Y-27632 had reduced TRPM6 expression levels in all cell lines. Y-27632 increased the expression levels of TRPC7 in all cell lines. In conclusion, this is the first study demonstrating that the inhibition of ROCK pathway changes the expression levels of some TRP genes. Also, our study has firstly shown that the expression levels of the TRP genes which are suggested as a diagnostic and prognostic biomarker in BC, were changed with the treatment of fasudil and Y-27632. PMID:24839003

  16. Vitamin D receptor expression levels determine the severity and complexity of disease progression among leprosy reaction patients

    PubMed Central

    Mandal, D.; Reja, A.H.H.; Biswas, N.; Bhattacharyya, P.; Patra, P.K.; Bhattacharya, B.

    2015-01-01

    We studied the roles of vitamin D and its receptor, VDR, in the progression of leprosy. The majority of individuals with leprosy from Kolkata, India, with a type 1 or type 2 reaction have low levels of vitamin D3 in serum samples. Interestingly, individuals with a type 2 reaction associated with neuritis/erythema nodosum leprosum had very low VDR mRNA expression levels, ranging from 5% to 10%, compared to that of healthy control subjects; these patients also had a high bacilli index, ranging from 3+ to 5+. This is the first report to indicate that VDR expression levels may determine the complexity and severity of the progression of leprosy. PMID:26106480

  17. Vitamin D receptor expression levels determine the severity and complexity of disease progression among leprosy reaction patients.

    PubMed

    Mandal, D; Reja, A H H; Biswas, N; Bhattacharyya, P; Patra, P K; Bhattacharya, B

    2015-07-01

    We studied the roles of vitamin D and its receptor, VDR, in the progression of leprosy. The majority of individuals with leprosy from Kolkata, India, with a type 1 or type 2 reaction have low levels of vitamin D3 in serum samples. Interestingly, individuals with a type 2 reaction associated with neuritis/erythema nodosum leprosum had very low VDR mRNA expression levels, ranging from 5% to 10%, compared to that of healthy control subjects; these patients also had a high bacilli index, ranging from 3+ to 5+. This is the first report to indicate that VDR expression levels may determine the complexity and severity of the progression of leprosy. PMID:26106480

  18. Relationship between expression levels and atherogenesis in scavenger receptor Class B, Type I Transgenics

    SciTech Connect

    Ueda, Yukihiko; Gong, Elaine; Royer, Lori; Cooper, Philip N.; Francone, Omar L; Rubin, Edward M.

    2000-03-15

    Both in vitro and in vivo studies of SR-BI have implicated it as a likely participant in the metabolism of HDL cholesterol. To investigate SR-BI's effect on atherogenesis we examined two lines of SR-BI transgenic mice with high (10-fold increases) and low (2-fold increases) in SR-BI expression in an inbred mouse background hemizygous for a human apo B transgene. Unlike non-HDL cholesterol levels which minimally differed in the various groups of animals, HDL cholesterol levels were inversely related to SR-BI expression. Mice with the low expression SR-BI transgene had a 50% reduction in HDL cholesterol while the high expression SR-BI transgene was associated with two-fold decreases in HDL as well as dramatic alterations in HDL composition and size including the near absence of a-migrating particles as determined by 2-dimensional electrophoresis. The low expression SR-BI/apo B transgenics had more than a two-fold decrease in the development of diet induced fatty streak lesions compared t o the apo B transgenics (4448{+-}1908 {mu}m2/aorta to 10133 {+-} 4035 {mu}m2/aorta; p<0.001), while the high expression SR-BI/apo B transgenics had an atherogenic response similar to that of the apo B transgenics (14692{+-}7238 {mu}m2/aorta) but three-fold greater than the low SR-BI/apo B mice (p<0.001). The prominent anti-atherogenic effect of moderate SR-BI expression provides in vivo support for the hypothesis that HDL functions to inhibit atherogenesis through its interactions with SR-BI in facilitating reverse cholesterol transport. The failure of the high SR-BI/apo B transgenics to have similar or even greater reductions in atherogenesis suggests that the changes resulting from extremely high SR-BI expression including dramatic changes in lipoproteins may have both pro- and anti-atherogenic consequences illustrating the complexity of the relationship between SR-BI and atherogenesis.

  19. Prostacyclin receptor expression on platelets of humans with type 2 diabetes is inversely correlated with hemoglobin A1c levels.

    PubMed

    Knebel, Stephanie M; Sprague, Randy S; Stephenson, Alan H

    2015-01-01

    Inappropriate platelet aggregation can result in thrombosis and tissue ischemia. When compared to healthy human platelets, those of humans with type 2 diabetes (DM2) exhibit increased aggregation when stimulated. Activation of the platelet prostacyclin receptor (IPR) results in cAMP accumulation and inhibition of platelet aggregation. We hypothesized that DM2 platelets express decreased IPR when compared to platelets of healthy humans, resulting in decreased IPR agonist-induced cAMP accumulation. We measured IPR expression with radioligand binding of [(3)H]-iloprost, a stable prostacyclin analog, and with Western blotting of the IPR protein. Iloprost-stimulated platelet cAMP levels were used to identify the functional response to IPR activation. IPR binding, expression of the IPR protein and the levels of cAMP in platelets incubated with iloprost were significantly decreased in DM2 platelets when compared to platelets of healthy humans. IPR expression decreased in platelets as glycemic control of the subjects worsened, as indicated by increased hemoglobin A1c levels. Taken together, these findings suggest that reduced IPR expression in DM2 platelets may contribute to platelet hyperactivity in humans with type 2 diabetes. PMID:25617843

  20. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs

    PubMed Central

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  1. Neuromedin B and Its Receptor: Gene Cloning, Tissue Distribution and Expression Levels of the Reproductive Axis in Pigs.

    PubMed

    Ma, Zhiyu; Su, Juan; Guo, Tingting; Jin, Mengmeng; Li, Xiang; Lei, Zhihai; Hou, Yuanlong; Li, Xiaoliang; Jia, Cuicui; Zhang, Zheng; Ahmed, Ejlal

    2016-01-01

    Neuromedin B is one member of a family of bombesin-like peptides, which performs a variety of physiological functions via their receptor (NMBR) in most mammals. However, the genes encoding NMB and NMBR and their functions especially reproduction of the pigs are currently not fully understood. To research the physiological functions of NMB, we cloned and analyzed the NMB and NMBR genes, and systematically investigated the expression levels of NMB and NMBR mRNA using relative real-time PCR and the distribution of NMBR by immunohistochemistry (IHC). Experimental results show that the sequences of the amino acid and gene of NMB and NMBR were highly conservative and homology in many species, Significantly, the relative RT-PCR results revealed that NMB was mainly expressed in the central nervous system (CNS), whereas NMBR is highly expressed in peripheral tissues and organs, such as endocrine tissues, glands and reproductive organs. The IHC results show that NMBR positive cells were widely distributed in the body, such as respiratory and circulatory system, digestive system, urogenital system, in lymphatic organs and in the endocrine system. We also systematically investigated expression levels of NMB and NMBR in the reproductive axis using relative real-time PCR. In sow estrous cycle, the hypothalamic levels of both NMB and NMBR mRAN were similar, but the expression levels of the pituitary were negatively correlated. Expression levels in the ovarian system are lowest in metestrus phases and highest in proestrus and estrus phases. In boar post-natal development stages, the hypothalamic, pituitary and testicular levels of NMB and NMBR mRNAs showed developmental changes on postnatal day 30, 60, 90 and 120. Taken together, this study provided molecular and morphological data necessary for further research of physiological function of NMB/NMBR system in the pigs. PMID:27010315

  2. Elevated Toll-Like Receptor-Induced CXCL8 Secretion in Human Blood Basophils from Allergic Donors Is Independent of Toll-Like Receptor Expression Levels

    PubMed Central

    Steiner, Markus; Hawranek, Thomas; Schneider, Michael; Ferreira, Fatima; Horejs-Hoeck, Jutta; Harrer, Andrea; Himly, Martin

    2016-01-01

    Human blood basophils have recently gained interest in addition to their function as allergic effector cells. Previous work suggests the involvement of innate immune mechanisms in the development and exacerbation of allergic responses, which might be mediated by basophils. We assayed the expression levels of Toll-like receptor (TLR) 1, 2, 4 and 6 on purified basophils from birch pollen-, house dust mite-, and non-allergic individuals. Additionally, we compared cytokine and chemokine secretion upon TLR stimulation in these basophil donor groups. Expression of TLR4 on the basophils of the allergic donor groups was decreased and CXCL8 secretion was elevated upon stimulation of TLR1/2 and TLR2/6 compared to the non-allergic donors. Decreased TLR expression and elevated CXCL8 secretion may represent possible mechanisms for aggravation of allergic symptoms in case of parasitic infection. PMID:26870962

  3. The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2.

    PubMed

    Jäger, Michael; Schoberth, Alexandra; Ruf, Peter; Hess, Jürgen; Lindhofer, Horst

    2009-05-15

    Human epidermal growth factor receptor 2 (HER2/neu) is an important target for the treatment of the breast cancers in which it is overexpressed. However, no approved anti-HER2/neu therapy is available for the majority of breast cancer patients, who express HER2/neu at low levels (with scores of 1+ or 2+/fluorescence in situ hybridization-negative). The trifunctional antibody ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and accessory cells form to cause tumor cell lysis. In a phase I trial with metastatic breast cancer patients, ertumaxomab could be applied safely and resulted in radiographically confirmed clinical responses. In this study, we compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that express HER2/neu at low and high levels. Under optimal conditions for trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only ertumaxomab was able to mediate the elimination of tumor cell lines that express HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a Th1-based cytokine release, a unique mode of action of trifunctional antibodies. Competitive binding studies with trastuzumab and 520C9 mapped the binding site of ertumaxomab to the extracellular regions II and III of the HER2/neu ectodomain. This site is distinct from the binding site of trastuzumab, so that HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence of high amounts of trastuzumab. The ability of ertumaxomab to induce cytotoxicity against various tumor cell lines, including those with low HER2/neu antigen density, may provide a novel therapeutic option for breast cancer patients who are not eligible for trastuzumab treatment. PMID:19435924

  4. Extracellular glutamate level and NMDA receptor subunit expression in mouse olfactory bulb following nanoparticle-rich diesel exhaust exposure.

    PubMed

    Win-Shwe, Tin-Tin; Mitsushima, Dai; Yamamoto, Shoji; Fujitani, Yuji; Funabashi, Toshiya; Hirano, Seishiro; Fujimaki, Hidekazu

    2009-08-01

    In this present study, we aimed to investigate the extracellular glutamate level and memory function-related gene expression in the mouse olfactory bulb after exposure of the animals to nanoparticle-rich diesel exhaust (NRDE) with or without bacterial cell wall component. Lipoteichoic acid (LTA), a cell wall component derived from Staphylococcus aureus, was used to induce systemic inflammation. Male BALB/c mice were exposed to clean air (particle concentration, 4.58 microg/m(3)) or NRDE (148.86 microg/m(3)) 5 h per day on 5 consecutive days of the week for 4 wk with or without weekly intraperitoneal injection of LTA. We examined the extracellular glutamate levels in the olfactory bulb using in vivo microdialysis and high-performance liquid chromatography assay. Then, we collected the olfactory bulb to examine the expression of N-methyl-D-aspartate (NMDA) receptor subunits (NR1, NR2A, and NR2B) and calcium/calmodulin-dependent protein kinase (CaMK) IV and cyclic AMP response element binding protein (CREB)-1 using real-time reverse-transcription polymerase chain reaction (RT-PCR). NRDE and/or LTA caused significantly increased extracellular glutamate levels in the olfactory bulb of mice. Moreover, the exposure of mice to NRDE upregulates NR1, NR2A, NR2B, and CaMKIV mRNAs in the olfactory bulb, while LTA upregulates only NR2B and CREB1 mRNAs. These findings suggest that NRDE and LTA cause glutamate-induced neurotoxicity separately and accompanied by changes in the expression of NMDA receptor subunits and related kinase and transcription factor in the mouse olfactory bulb. This is the first study to show the correlation between glutamate toxicity and memory function-related gene expressions in the mouse olfactory bulb following exposure to NRDE. PMID:19653804

  5. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment.

    PubMed

    Sustarsic, Elahu G; Junnila, Riia K; Kopchick, John J

    2013-11-01

    Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute's NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  6. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    PubMed Central

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-01-01

    Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on this data, GH could be a new therapeutic target in melanoma. PMID:24134847

  7. MicroRNA-148a regulates LDL receptor and ABCA1 expression to control circulating lipoprotein levels.

    PubMed

    Goedeke, Leigh; Rotllan, Noemi; Canfrán-Duque, Alberto; Aranda, Juan F; Ramírez, Cristina M; Araldi, Elisa; Lin, Chin-Sheng; Anderson, Norma N; Wagschal, Alexandre; de Cabo, Rafael; Horton, Jay D; Lasunción, Miguel A; Näär, Anders M; Suárez, Yajaira; Fernández-Hernando, Carlos

    2015-11-01

    The hepatic low-density lipoprotein receptor (LDLR) pathway is essential for clearing circulating LDL cholesterol (LDL-C). Whereas the transcriptional regulation of LDLR is well characterized, the post-transcriptional mechanisms that govern LDLR expression are just beginning to emerge. Here we develop a high-throughput genome-wide screening assay to systematically identify microRNAs (miRNAs) that regulate LDLR activity in human hepatic cells. From this screen we identified and characterized miR-148a as a negative regulator of LDLR expression and activity and defined a sterol regulatory element-binding protein 1 (SREBP1)-mediated pathway through which miR-148a regulates LDL-C uptake. In mice, inhibition of miR-148a increased hepatic LDLR expression and decreased plasma LDL-C. Moreover, we found that miR-148a regulates hepatic expression of ATP-binding cassette, subfamily A, member 1 (ABCA1) and circulating high-density lipoprotein cholesterol (HDL-C) levels in vivo. These studies uncover a role for miR-148a as a key regulator of hepatic LDL-C clearance through direct modulation of LDLR expression and demonstrate the therapeutic potential of inhibiting miR-148a to ameliorate an elevated LDL-C/HDL-C ratio, a prominent risk factor for cardiovascular disease. PMID:26437365

  8. High-level expression in Saccharomyces cerevisiae enables isolation and spectroscopic characterization of functional human adenosine A2a receptor.

    PubMed

    O'Malley, Michelle A; Lazarova, Tzvetana; Britton, Zachary T; Robinson, Anne S

    2007-08-01

    The G-protein coupled receptors (GPCRs) are a class of membrane proteins that trigger cellular responses to external stimuli, and are believed to be targets for nearly half of all pharmaceutical drugs on the market. However, little is known regarding their folding and cellular interactions, as well as what factors are crucial for their activity. Further structural characterization of GPCRs has largely been complicated by problems with expression, purification, and preservation of activity in vitro. Previously, we have demonstrated high-level expression (approximately 4mg/L of culture) of functional human adenosine A(2)a receptor fused to a green fluorescent protein (A(2)aR-GFP) from Saccharomyces cerevisiae. In this work, we re-engineered A(2)aR with a purification tag, developed an adequate purification scheme, and performed biophysical characterization on purified receptors. Milligram amounts per liter of culture of A(2)aR and A(2)aR-GFP were functionally expressed in S. cerevisiae, with a C-terminal deca-histidine tag. Lysis procedures were developed for optimal membrane protein solubilization and recovery through monitoring fluorescence of A(2)aR-GFP-His(10). One-step purification of the protein was achieved through immobilized metal affinity chromatography. After initial solubilization in n-dodecyl-beta-d-maltoside (DDM), a combination of added cholesterol hemisuccinate (CHS) in 3-(3-cholamidopropyl)-dimethylammoniopropane sulfonate (CHAPS) was required to stabilize the functional state of the protein. Isolated A(2)aR under these conditions was found to be largely alpha-helical, and properly incorporated into a mixed-micelle environment. The A(2)a-His(10) receptor was purified in quantities of 6+/-2mg/L of culture, with ligand-binding yields of 1mg/L, although all protein bound to xanthine affinity resin. This represents the highest purified total and functional yields for A(2)aR yet achieved from any heterologous expression system. PMID:17591446

  9. Human metastatic melanoma cell lines express high levels of growth hormone receptor and respond to GH treatment

    SciTech Connect

    Sustarsic, Elahu G.; Junnila, Riia K.; Kopchick, John J.

    2013-11-08

    Highlights: •Most cancer types of the NCI60 have sub-sets of cell lines with high GHR expression. •GHR is highly expressed in melanoma cell lines. •GHR is elevated in advanced stage IV metastatic tumors vs. stage III. •GH treatment of metastatic melanoma cell lines alters growth and cell signaling. -- Abstract: Accumulating evidence implicates the growth hormone receptor (GHR) in carcinogenesis. While multiple studies show evidence for expression of growth hormone (GH) and GHR mRNA in human cancer tissue, there is a lack of quantification and only a few cancer types have been investigated. The National Cancer Institute’s NCI60 panel includes 60 cancer cell lines from nine types of human cancer: breast, CNS, colon, leukemia, melanoma, non-small cell lung, ovarian, prostate and renal. We utilized this panel to quantify expression of GHR, GH, prolactin receptor (PRLR) and prolactin (PRL) mRNA with real-time RT qPCR. Both GHR and PRLR show a broad range of expression within and among most cancer types. Strikingly, GHR expression is nearly 50-fold higher in melanoma than in the panel as a whole. Analysis of human metastatic melanoma biopsies confirmed GHR gene expression in melanoma tissue. In these human biopsies, the level of GHR mRNA is elevated in advanced stage IV tumor samples compared to stage III. Due to the novel finding of high GHR in melanoma, we examined the effect of GH treatment on three NCI60 melanoma lines (MDA-MB-435, UACC-62 and SK-MEL-5). GH increased proliferation in two out of three cell lines tested. Further analysis revealed GH-induced activation of STAT5 and mTOR in a cell line dependent manner. In conclusion, we have identified cell lines and cancer types that are ideal to study the role of GH and PRL in cancer, yet have been largely overlooked. Furthermore, we found that human metastatic melanoma tumors express GHR and cell lines possess active GHRs that can modulate multiple signaling pathways and alter cell proliferation. Based on

  10. Expression levels of estrogen receptor α mRNA in peripheral blood cells are an independent biomarker for postmenopausal osteoporosis☆

    PubMed Central

    Chou, Chi-Wen; Chiang, Tsay-I; Chang, I-Chang; Huang, Chung-Hung; Cheng, Ya-Wen

    2016-01-01

    Background The up- and down-regulation of the osteoclastogenesis response depends on the estrogen/estrogen receptor (ER) signaling pathway. Previous reports have shown that the promoter hypermethylation and gene polymorphism of ERα are risks for menopausal osteoporosis. No previous study has evaluated the expression levels of ERα mRNA in menopausal osteoporosis using human subjects. We hypothesized that ERα mRNA expression may show less resistance to postmenopausal osteoporosis. Methods In this study, we enrolled 107 women older than 45 years without menstruation and classified them into control, osteopenia, and osteoporosis groups depending on their T-scores. The ERα mRNA levels in peripheral blood cells (PBCs) were analyzed via quantitative real-time reverse-transcription polymerase chain reaction (QRT-PCR), and estrogen in the serum was detected via ELISA. Results ERα mRNA levels in PBCs had a negative correlation with age and a positive correlation with estrogen and BAP in the osteopenia and osteoporosis groups, but not in the control group. Additionally, multivariate analysis showed that older age (> 55 years), and low ERα mRNA levels in PBLs (≦ 250.39 copies/μg DNA) were associated with an approximately 9.188-, and 31.25-fold risk of osteoporosis. Conclusion We conclude that ERα mRNA levels in PBLs could be used as an independent risk factor for postmenopausal osteoporosis. General significance Our findings suggested that ERα mRNA levels in PBLs may be more important than age and serum estrogen levels. PMID:27051599

  11. Combined effects of levonorgestrel and quinestrol on reproductive hormone levels and receptor expression in females of the Mongolian gerbil (Meriones unguiculatus).

    PubMed

    Lv, Xiaohui; Shi, Dazhao

    2012-01-01

    The effects of treatment with a combination of levonorgestrel and quinestrol (EP-1; ratio of 2:1) on reproductive hormone levels and the expression of their receptors in female Mongolian gerbils were examined. We show that serum follicle-stimulating hormone (FSH) and luteinizing hormone (LH) decreased, whereas serum estradiol (E2) and progesterone (P4) increased after EP-1 treatment. EP1 down-regulated mRNA expression of the follicle-stimulating hormone receptor (FSHR) and the estrogen receptor (ER) βin the ovary. EP-1 up-regulated the mRNA expression of the luteinizing hormone receptor (LHR) and the progesterone receptor (PR) in the ovary as well as ERα and PR in the uterus of Mongolian gerbils. The effects were time-dependent and dose-dependent. EP-1 had no obvious effects on ERα mRNA expression in the ovary. The current study demonstrates that the effect of EP-1 on the expression of ER subtypes is tissue-specific in Mongolian gerbils. EP-1 disrupted the reproductive endocrinology of the Mongolian gerbil. These findings suggest that the effects of EP-1 on reproductive hormone levels and their receptor expression in Mongolian gerbils may be the result of synergistic actions of levonorgestrel and quinestrol, with quinestrol playing the major role. PMID:22233494

  12. RTN3 Regulates the Expression Level of Chemokine Receptor CXCR4 and is Required for Migration of Primordial Germ Cells

    PubMed Central

    Li, Haitao; Liang, Rong; Lu, Yanan; Wang, Mengxia; Li, Zandong

    2016-01-01

    CXCR4 is a crucial chemokine receptor that plays key roles in primordial germ cell (PGC) homing. To further characterize the CXCR4-mediated migration of PGCs, we screened CXCR4-interacting proteins using yeast two-hybrid screening. We identified reticulon3 (RTN3), a member of the reticulon family, and considered an apoptotic signal transducer, as able to interact directly with CXCR4. Furthermore, we discovered that the mRNA and protein expression levels of CXCR4 could be regulated by RTN3. We also found that RTN3 altered CXCR4 translocation and localization. Moreover, increasing the signaling of either CXCR4b or RTN3 produced similar PGC mislocalization phenotypes in zebrafish. These results suggested that RTN3 modulates PGC migration through interaction with, and regulation of, CXCR4. PMID:27070582

  13. Vitamin D3 supplementation increases insulin level by regulating altered IP3 and AMPA receptor expression in the pancreatic islets of streptozotocin-induced diabetic rat.

    PubMed

    Jayanarayanan, Sadanandan; Anju, Thoppil R; Smijin, Soman; Paulose, Cheramadathikudiyil Skaria

    2015-10-01

    Pancreatic islets, particularly insulin-secreting β cells, share common characteristics with neurons. Glutamate is one of the major excitatory neurotransmitter in the brain and pancreas, and its action is mediated through glutamate receptors. In the present work, we analysed the role of vitamin D3 in the modulation of AMPA receptor subunit and their functional role in insulin release. Radio receptor binding study in diabetic rats showed a significant increase in AMPA receptor density. Insulin AMPA colabelling study showed an altered AMPA GluR2 and GluR4 subunit expression in the pancreatic beta cells. We also found lowered IP3 content and decreased IP3 receptor in pancreas of diabetic rats. The alterations in AMPA and IP3 receptor resulted in reduced cytosolic calcium level concentration, which further blocks Ca(2+)-mediated insulin release. Vitamin D3 supplementation restored the alteration in vitamin D receptor expression, AMPA receptor density and AMPA and IP3 receptor expression in the pancreatic islets that helps to restore the calcium-mediated insulin secretion. Our study reveals the antidiabetic property of vitamin D3 that is suggested to have therapeutic role through regulating glutamatergic function in diabetic rats. PMID:26054778

  14. Serum level of soluble interleukin-2 receptor correlates with CD25 expression in patients with T lymphoblastic lymphoma.

    PubMed

    Toji, Tomohiro; Takata, Katsuyoshi; Sato, Yasuharu; Miyata-Takata, Tomoko; Hayashi, Eiko; Habara, Toshiyuki; Maeda, Yoshinobu; Tanimoto, Mitsune; Yoshino, Tadashi

    2015-08-01

    Acute lymphoblastic leukaemia/lymphoma (ALL/LBL) is an aggressive form of non-Hodgkin's lymphoma (NHL) affecting B-cells or T-cells, respectively. The serum level of soluble interleukin-2 receptor (sIL-2R) is known to reflect the immune activity and tumour volume in aggressive NHL; however, the release of sIL-2R in LBL has not been extensively studied. Further, the relationship between sIL-2R release and the expression level of IL-2R α subunit (CD25) remains unknown. In the present study, we examined the serum level of sIL-2R in 23 patients with T lymphoblactic lymphoma (T-LBL) and compared these with the levels in 20 patient with T acute lymphoblastic leukaemia (T-ALL), 40 patients with diffuse large B-cell lymphoma (DLBCL) and 40 patients with peripheral T-cell lymphoma (PTCL), not otherwise specified. The release of sIL-2R into the serum in patients with T-LBL was significantly lower than that for T-ALL, DLBCL and PTCL (p<0.001). Immunohistochemistry revealed that CD25 expression was correlated with the serum level of sIL-2R in T-LBL (p=0.0069), whereas no correlation was found to exist between serum sIL-2R levels and CD25 expression in patients with DLBCL (p=0.348) and PTCL (p=0.266). Furthermore, double immunohistochemical analysis revealed that CD25-positive cells were also found to be Foxp3-positive non-neoplastic T-cells. In conclusion, CD25-positive non-neoplastic T-cells in T-LBL are presumed to be the primary source of sIL-2R, and the low number of cells present results in a lower level of sIL-2R released into the serum compared with the other aggressive and highly aggressive lymphomas. PMID:25935549

  15. Dexamethasone inhibits human interleukin 2 but not interleukin 2 receptor gene expression in vitro at the level of nuclear transcription.

    PubMed Central

    Boumpas, D T; Anastassiou, E D; Older, S A; Tsokos, G C; Nelson, D L; Balow, J E

    1991-01-01

    Glucocorticosteroids have an inhibitory effect on the expression of interleukin 2 (IL-2) and interleukin 2 receptor (IL-2R) genes. To determine the mechanisms of this inhibition, human T lymphocytes were stimulated with mitogens in the presence of dexamethasone. Nuclear transcription run-off assays showed that high doses of dexamethasone inhibited the transcription of the IL-2 gene but not that of the IL-2R gene. Post-transcriptionally, high doses of dexamethasone (10(-4) M) were required to inhibit IL-2R mRNA levels by 50%, whereas lower doses (10(-6) M) inhibited by greater than 70% the accumulation of IL-2 mRNA. IL-2 mRNA half-life decreased in the presence of dexamethasone (10(-6) M) by approximately 50%. At the protein product level, dexamethasone inhibited both IL-2 production, as well as cell surface and soluble forms of IL-2R. IL-2R gene expression was inhibited for at least 72 h after exposure of cells to dexamethasone. In the presence of exogenous IL-2, dexamethasone failed to exert a significant effect on the production of IL-2R protein. These data indicate that dexamethasone has a greater effect on the expression of the IL-2 gene than on the IL-2R gene. Dexamethasone both inhibits transcription of the IL-2 gene and decreases the stability of IL-2 mRNA. The effect of dexamethasone on the IL-2R gene is post-transcriptional and may result indirectly from decreased IL-2 production. Images PMID:2022743

  16. Effect of a povidone-iodine intrauterine infusion on progesterone levels and endometrial steroid receptor expression in mares

    PubMed Central

    2010-01-01

    Background Intrauterine infusions have been widely used for the treatment of endometritis in the mare. Nevertheless, their consequences on endocrine and endometrial molecular aspects are unknown. We studied the effect of a 1% povidone-iodine solution intrauterine infusion on progesterone levels, endometrial histology and estrogen (ERα) and progesterone (PR) receptor distribution by immunohistochemistry. Methods Fourteen healthy mares were used in this study. Estruses were synchronized and seven mares were treated with intrauterine infusions at days 0 and 2 post ovulation of two consecutive estrous cycles. Uterine biopsy samples were taken on days 6 and 15 post ovulation. Results The treatment did not induce an inflammatory response indicating endometritis, neither affected the ERα. However, it reduced the percentage of PR positive cells (PPC) on day 6 (deep glandular epithelium, control: 95.7 vs. infused: 61.5, P < 0.05). Treated mares tended to have lower progesterone levels on day 2 (3.9 ng/ml vs. 6.6 ng/ml, P = 0.07), and higher levels on day 15 compared with controls (4.4 ng/ml vs. 1.3 ng/ml, P = 0.07). Conclusion a 1% povidone-iodine infusion during days 0 and 2 post ovulation in healthy mares did not induce histological changes indicating endometritis, but altered progesterone concentrations and reduced the expression of endometrial PR at day 6 without affecting the ERα. These changes could reduce embryo survival. PMID:21162724

  17. Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

    SciTech Connect

    Gonda, Kohsuke; Miyashita, Minoru; Watanabe, Mika; Takahashi, Yayoi; Goda, Hideki; Okada, Hisatake; Nakano, Yasushi; Tada, Hiroshi; Amari, Masakazu; Ohuchi, Noriaki

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Organic fluorescent material-assembled nanoparticles for IHC were prepared. Black-Right-Pointing-Pointer New nanoparticle fluorescent intensity was 10.2-fold greater than Qdot655. Black-Right-Pointing-Pointer Nanoparticle staining analyzed a wide range of ER expression levels in tissue. Black-Right-Pointing-Pointer Nanoparticle staining enhanced the quantitative sensitivity for ER diagnosis. -- Abstract: The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3 Prime -diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to

  18. Spontaneous cell fusion in macrophage cultures expressing high levels of the P2Z/P2X7 receptor.

    PubMed

    Chiozzi, P; Sanz, J M; Ferrari, D; Falzoni, S; Aleotti, A; Buell, G N; Collo, G; Di Virgilio, F

    1997-08-11

    Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X7. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X7 receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207- 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X7 receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X7 blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X7 receptor is involved in macrophage fusion. PMID:9245796

  19. Membrane-tethered AKT kinase regulates basal synaptic transmission and early phase LTP expression by modulation of post-synaptic AMPA receptor level.

    PubMed

    Pen, Y; Borovok, N; Reichenstein, M; Sheinin, A; Michaelevski, I

    2016-09-01

    The serine/threonine kinase AKT/PKB plays a fundamental role in a wide variety of neuronal functions, including neuronal cell development, axonal growth, and synaptic plasticity. Multiple evidence link AKT signaling pathways to regulation of late phase long-term synaptic plasticity, synaptogenesis, and spinogenesis, as well as long-term memory formation. Nevertheless, the downstream effectors mediating the effects of AKT on early phase long-term potentiation (eLTP) are currently unknown. Here we report that using different regimes of pharmacological activation and inhibition of AKT activity in acute hippocampal slices, we found that AKT regulates the post-synaptic expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPA) receptors affecting solely the expression of eLTP, with no effect on its induction and maintenance. We further show that both maintenance of basal synaptic activity and expression of eLTP require plasma membrane tethering by activated AKT and that basal synaptic activity may be regulated via the direct effects of AKT1 on the expression level of post-synaptic AMPA receptors bypassing the canonical AKT signaling. Finally, we establish that eLTP expression requires the involvement of both the canonical AKT signaling pathways and the direct effect of AKT1 on AMPA receptor activity/expression in the post-synaptic membrane. © 2016 Wiley Periodicals, Inc. PMID:27068236

  20. Monocyte surface expression of Fcγ receptor RI (CD64), a biomarker reflecting type-I interferon levels in systemic lupus erythematosus

    PubMed Central

    2010-01-01

    Introduction More than half of systemic lupus erythematosus (SLE) patients show evidence of excess type I interferon (IFN-I) production, a phenotype associated with renal disease and certain autoantibodies. However, detection of IFN-I proteins in serum is unreliable, and the measurement of interferon-stimulated gene (ISG) expression is expensive and time consuming. The aim of this study was to identify a surrogate marker for IFN-I activity in clinical samples for monitoring disease activity and response to therapy. Methods Monocyte surface expression of Fcγ receptors (FcγRs), chemokine receptors, and activation markers were analyzed with flow cytometry in whole blood from patients with SLE and healthy controls. FcγR expression also was measured in peripheral blood mononuclear cells (PBMCs) from healthy controls cultured with Toll-like receptor (TLR) agonists, cytokines, or serum from SLE patients. Expression of ISGs was analyzed with real-time PCR. Results Circulating CD14+ monocytes from SLE patients showed increased surface expression of FcγRI (CD64). The mean fluorescent intensity of CD64 staining correlated highly with the ISG expression (MX1, IFI44, and Ly6E). In vitro, IFN-I as well as TLR7 and TLR9 agonists, induced CD64 expression on monocytes from healthy controls. Exposure of monocytes from healthy controls to SLE sera also upregulated the expression of CD64 in an IFN-I-dependent manner. Decreased CD64 expression was observed concomitant with the reduction of ISG expression after high-dose corticosteroid therapy. Conclusions Expression of CD64 on circulating monocytes is IFN-I inducible and highly correlated with ISG expression. Flow-cytometry analysis of CD64 expression on circulating monocytes is a convenient and rapid approach for estimating IFN-I levels in SLE patients. PMID:20478071

  1. Expression of the survival of motor neuron (SMN) gene in primary neurons and increase in SMN levels by activation of the N-methyl-D-aspartate glutamate receptor.

    PubMed

    Andreassi, Catia; Patrizi, Anna Letizia; Monani, Umrao R; Burghes, A H M; Brahe, Christina; Eboli, Maria Luisa

    2002-03-01

    Spinal muscular atrophy (SMA) is a common motor neuron degenerative disease caused by mutations of the survival of motor neuron (SMN) gene. The SMN protein is expressed ubiquitously as part of a 300-kilodalton multi-protein complex, incorporating several proteins critically required in pre-mRNA splicing. Although SMN mutations render SMN defective in this role, the specific alpha-motor neuron degenerative phenotype seen in the disease remains unexplained. During the differentiation process of spinal motor neurons and cerebellar granule cells, the acquisition of mature electrophysiological and molecular properties is linked to the activation of the glutamate receptors of N-methyl-D-aspartate (NMDA) subtype. We have used primary cultures of rat cerebellar granules to study SMN expression during neuronal differentiation in vitro and in response to the activation of the NMDA receptor. We report that the expression of gems, the nuclear structures where SMN concentrates, is developmentally regulated. The highest expression is associated with the cell clustering phase and expression of NMDA receptors. Stimulation of the NMDA receptor induces an increase in gem number and in SMN transcription, through activation of its promoter. These results demonstrate that SMN levels are dependent on synaptic activity, implying that SMN may have important neuron-specific functions downstream of synaptic activation. PMID:12030329

  2. Expression of insect α6-like nicotinic acetylcholine receptors in Drosophila melanogaster highlights a high level of conservation of the receptor:spinosyn interaction.

    PubMed

    Perry, Trent; Somers, Jason; Yang, Ying Ting; Batterham, Philip

    2015-09-01

    Insecticide research has often relied on model species for elucidating the resistance mechanisms present in the targeted pests. The accuracy and applicability of extrapolations of these laboratory findings to field conditions varies but, for target site resistance, conserved mechanisms are generally the rule rather than the exception (Perry et al., 2011). The spinosyn class of insecticides appear to fit this paradigm and are a pest control option with many uses in both crop and animal protection. Resistance to spinosyns has been identified in both laboratory-selected and field-collected pest insects. Studies using the model insect, Drosophila melanogaster, have identified the nicotinic acetylcholine receptor subunit, Dα6 as an important target of the insecticide spinosad (Perry et al., 2007; Watson et al., 2010). Field-isolated resistant strains of several agricultural pest insects provide evidence that resistance cases are often associated with mutations in orthologues to Dα6 (Baxter et al., 2010; Puinean et al., 2013). The expression of these receptors is difficult in heterologous systems. In order to examine the biology of the Dα6 receptor subunit further, we used Drosophila as a model and developed an in vivo rescue system. This allowed us to express four different isoforms of Dα6 and show that each is able to rescue the response to spinosad. Regulatory sequences upstream of the Dα6 gene able to rescue the resistance phenotype were identified. Expression of other D. melanogaster subunits revealed that the rescue phenotype appears to be Dα6 specific. We also demonstrate that expression of pest insect orthologues of Dα6 from a variety of species are capable of rescuing the spinosad response phenotype, verifying the relevance of this receptor to resistance monitoring in the field. In the absence of a robust heterologous expression system, this study presents an in vivo model that will be useful in analysing many other aspects of these receptors and

  3. Application of NIR fluorescent markers to quantify expression level of HER2 receptors in carcinomas in vivo

    NASA Astrophysics Data System (ADS)

    Chernomordik, Victor; Hassan, Moinuddin; Lee, Sang Bong; Zielinski, Rafal; Capala, Jacek; Gandjbakhche, Amir

    2010-02-01

    HER2 overexpression has been associated with a poor prognosis and resistance to therapy in breast cancer patients. However, quantitative estimates of this important characteristic have been limited to ex vivo ELISA essays of tissue biopsies and/or PET. We develop a novel approach in optical imaging, involving specific probes, not interfering with the binding of the therapeutic agents, thus, excluding competition between therapy and imaging. Affibody-based molecular probes seem to be ideal for in vivo analysis of HER2 receptors using near-infrared optical imaging. Fluorescence intensity distributions, originating from specific markers in the tumor area, can reveal the corresponding fluorophore concentration. We use temporal changes of the signal from a contrast agent, conjugated with HER2-specific Affibody as a signature to monitor in vivo the receptors status in mice with different HER2 over-expressed tumor models. Kinetic model, incorporating saturation of the bound ligands in the tumor area due to HER2 receptor concentration, is suggested to analyze relationship between tumor cell characteristics, i.e., HER2 overexpression, obtained by traditional ("golden standard") ex vivo methods (ELISA), and parameters, estimated from the series of images in vivo. Observed correlation between these parameters and HER2 overexpression substantiates application of our approach to quantify HER2 concentration in vivo.

  4. Prepubertal castration-associated developmental changes in sigma-1 receptor gene expression levels regulate hippocampus area CA1 activity during adolescence.

    PubMed

    Moradpour, Farshad; Fathollahi, Yaghoub; Naghdi, Nasser; Hosseinmardi, Nargess; Javan, Mohammad

    2016-07-01

    The functional relevance of sigma-1 (σ1 ) receptor expression in the rat hippocampal CA1 during adolescence (i.e., 35-60 days old) was explored. A selective antagonist for the σ1 receptor subtype, BD-1047, was applied to study hippocampal long-term potentiation (LTP) and spatial learning performance. Changes in the expression of the σ1 receptor subtype and its function were compared between castrated and sham-castrated rats. Castration reduced the magnitude of both field excitatory postsynaptic potential (fEPSP)-LTP and population spike (PS)-LTP at 35 days (d). BD-1047 decreased PS-LTP in sham-castrated rats, whereas BD-1047 reversed the effect of castration on fEPSP-LTP at 35 d. In addition, BD1047 impaired spatial learning and augmented σ1 receptor mRNA levels in castrated rats at 35 d. Surprisingly, neither castration nor BD1047 had an effect on fEPSP-LTP and PS-LTP, spatial learning ability or gene expression levels at 45 d. Castration had no effect on fEPSP-LTP but reduced PS-LTP at 60 d. BD1047 increased the magnitude of fEPSP-LTP, but had no effect on PS-LTP in castrated rats at 60 d. However, BD1047 reduced spatial learning ability, and σ1 receptor mRNA levels were decreased in castrated rats at 60 d. This study shows that σ1 receptors play a role in the regulation of both CA1 synaptic efficacy and spatial learning performance. The regulatory role of σ1 receptors in activity-dependent CA1-LTP is locality- and age-dependent, whereas its role in spatial learning ability is only age-dependent. Prepubertal castration-associated changes in the expression and function of the σ1 receptor during adolescence may play a developmental role in the regulation of hippocampal area CA1 activity and plasticity. © 2016 Wiley Periodicals, Inc. PMID:26860755

  5. Cadmium-induced decrement of the LH receptor expression and cAMP levels in the testis of rats.

    PubMed

    Gunnarsson, David; Nordberg, Gunnar; Lundgren, Per; Selstam, Gunnar

    2003-02-01

    Cadmium (Cd) is a widespread environmental pollutant, characterized by its ability to affect various organs. Adverse effect of Cd on the testis including decreased testosterone production are well-known phenomena, but the cellular events explaining these effects have not yet been established. In the present study the initial steps of gonadotropin mediated testosterone biosynthesis were examined in vivo in rats, in relation to Cd dose and time after injection. In the dose-response experiment Male Sprague-Dawley rats received a single subcutaneous (s.c.) injection of CdCl(2) (1, 5 or 10 micromol/kg body weight) and were sacrificed 48 h after injection. A statistically significant decrease in luteinizing hormone (LH) receptor mRNA level in the testicular tissue was demonstrated at the highest dose (10 micromol/kg). In the temporal-response experiment rats were given 10 micromol/kg of CdCl(2) s.c. and sacrificed 0.48, 4.8, 48 or 144 h after injection. LH receptor mRNA levels as well as cyclic adenosine monophosphate (cAMP) levels were found to be significantly lowered at 48 and 144 h. These observations of the mechanisms whereby Cd exerts its effect on the initial steps of testosterone biosynthesis are the first from in vivo experiments. PMID:12504342

  6. Effects of high levels of glucose on the steroidogenesis and the expression of adiponectin receptors in rat ovarian cells

    PubMed Central

    Chabrolle, Christine; JeanPierre, Eric; Tosca, Lucie; Ramé, Christelle; Dupont, Joëlle

    2008-01-01

    Background Reproductive dysfunction in the diabetic female rat is associated with altered folliculogenesis and steroidogenesis. However, the molecular mechanisms involved in the reduction of steroid production have not been described. Adiponectin is an adipocytokine that has insulin-sensitizing actions including stimulation of glucose uptake in muscle and suppression of glucose production in liver. Adiponectin acts via two receptor isoforms – AdipoR1 and AdipoR2 – that are regulated by hyperglycaemia and hyperinsulinaemia in liver and muscle. We have recently identified AdipoR1 and AdipoR2 in rat ovary. However, their regulation in ovaries of diabetic female rat remains to be elucidated. Methods We incubated rat primary granulosa cells in vitro with high concentrations of glucose (5 or 10 g/l) + or - FSH (10-8 M) or IGF-1 (10-8 M), and we studied the ovaries of streptozotocin-induced diabetic rats (STZ) in vivo. The levels of oestradiol and progesterone in culture medium and serum were measured by RIA. We used immunoblotting to assay key steroidogenesis factors (3beta HSD, p450scc, p450 aromatase, StAR), and adiponectin receptors and various elements of signalling pathways (MAPK ERK1/2 and AMPK) in vivo and in vitro. We also determined cell proliferation by [3H] thymidine incorporation. Results Glucose (5 or 10 g/l) impaired the in vitro production in rat granulosa cells of both progesterone and oestradiol in the basal state and in response to FSH and IGF-1 without affecting cell proliferation and viability. This was associated with substantial reductions in the amounts of 3beta HSD, p450scc, p450 aromatase and StAR proteins and MAPK ERK1/2 phosphorylation. In contrast, glucose did not affect the abundance of AdipoR1 or AdipoR2 proteins. In vivo, as expected, STZ treatment of rats caused hyperglycaemia and insulin, adiponectin and resistin deficiencies. Plasma progesterone and oestradiol levels were also reduced in STZ rats. However, the amounts of 3beta HSD

  7. Monoallelic Expression of Olfactory Receptors

    PubMed Central

    Monahan, Kevin; Lomvardas, Stavros

    2016-01-01

    The sense of smell collects vital information about the environment by detecting a multitude of chemical odorants. Breadth and sensitivity are provided by a huge number of chemosensory receptor proteins, including more than 1,400 olfactory receptors (ORs). Organizing the sensory information generated by these receptors so that it can be processed and evaluated by the central nervous system is a major challenge. This challenge is overcome by monogenic and monoallelic expression of OR genes. The single OR expressed by each olfactory sensory neuron determines the neuron’s odor sensitivity and the axonal connections it will make to downstream neurons in the olfactory bulb. The expression of a single OR per neuron is accomplished by coupling a slow chromatin-mediated activation process to a fast negative-feedback signal that prevents activation of additional ORs. Singular OR activation is likely orchestrated by a network of interchromosomal enhancer interactions and large-scale changes in nuclear architecture. PMID:26359778

  8. Ulipristal Acetate Antagonizes the Inhibitory Effect of Progesterone on Ciliary Beat Frequency and Upregulates Steroid Receptor Expression Levels in Human Fallopian Tubes.

    PubMed

    Yuan, Jiangjing; Zhao, Weihong; Yan, Mingxing; Zhu, Qian; Qin, Guojuan; Qiu, Jun; Zhang, Jian

    2015-12-01

    Ulipristal acetate (UPA) is a new selective progesterone receptor (PR) modulator used for emergency contraception. However, our understanding of its mechanisms of action on oviductal cilia is limited. The present study focused on the in vitro effects of UPA (0.1, 1, and 10 μmol/L) on the cilia and steroid receptors of human fallopian tubes. The ciliary beat frequency (CBF), the ultrastructure of cilia, and the levels of steroid receptors were measured. The effects of UPA on the progesterone-induced CBF reduction were also studied. Our results show that UPA dose dependently antagonizes the progesterone-induced CBF decrease, but it does not affect the CBF or the ultrastructure of the cilia. The UPA also upregulates the expression levels of the estrogen receptor α and the PR in the fallopian tubes. The results enable us to better understand the mechanisms by which UPA works as an emergency contraceptive and provides a scientific basis for its clinical application. PMID:26045548

  9. Chloral hydrate-dependent reduction in the peptidoglycan-induced inflammatory macrophage response is associated with lower expression levels of toll-like receptor 2

    PubMed Central

    PAN, QINGJUN; LIU, YUAN; ZHU, XUEZHI; LIU, HUAFENG

    2014-01-01

    The aim of this study was to investigate the effect and mechanism of action of chloral hydrate on the peptidoglycan (PGN)-induced inflammatory macrophage response. The effect of chloral hydrate on the production of tumor necrosis factor α (TNF-α) and interleukin-6 (IL-6) by murine peritoneal macrophages with PGN-stimulation was investigated. In addition, RAW264.7 cells transfected with a nuclear factor-κB (NF-κB) luciferase reporter plasmid stimulated by PGN were used to study the effect of chloral hydrate on the levels NF-κB activity. Flow cytometry and western blotting were performed to investigate the expression levels of toll-like receptor 2 (TLR2) in the treated RAW264.7 cells. It was identified that chloral hydrate reduced the levels of IL-6 and TNF-α produced by the peritoneal macrophages stimulated with PGN. The levels of NF-κB activity of the RAW264.7 cells stimulated by PGN decreased following treatment with chloral hydrate, which was associated with a reduction in the expression levels of TLR2 and reduced levels of TLR2 signal transduction. These data demonstrate that chloral hydrate reduced the magnitude of the PGN-induced inflammatory macrophage response associated with lower expression levels of TLR2. PMID:24940429

  10. Increased expression of mitochondrial benzodiazepine receptors following low-level light treatment facilitates enhanced protoporphyrin IX production in glioma-derived cells in vitro

    NASA Astrophysics Data System (ADS)

    Bisland, S. K.; Hassanali, N. S.; Johnson, C.; Wilson, B. C.

    2007-02-01

    This study investigates whether low level light treatment (LLLT) can enhance the expression of Peripheral-type mitochondrial benzodiazepine receptors (PBRs) on the glioma-derived tumour cell line, CNS-1, and by doing so promote the synthesis of protoporphyrin IX (PpIX) and increase the photodynamic therapy (PDT)-induced cell kill using 5-aminolevulinic acid (ALA). The endogenous photosensitizer, (PpIX) and related metabolites including coproporphyrin III are known to traffic via the PBRs on the outer mitochondrial membrane on their passage into or out of the mitochondria. Astrocyte-derived cells within the brain express PBRs, while neurons express the central-type of benzodiazepine receptor. CNS-1 cells were exposed to a range of differing low-level light protocols immediately prior to PDT. LLLT involved using broad-spectrum light or monochromatic laser light specific to 635 or 905 nm wavelength. Cells (5μ10 5) were exposed to a range of LLLT doses (0, 1 or 5 J/cm2) using a fixed intensity of 10 mW/cm2 and subsequently harvested for cell viability, immunofluorescence or western blot analysis of PBR expression. The amount of PpIX within the cells was determined using chemical extraction techniques. Results confirm the induction of PBR following LLLT is dependent on the dose and wavelength of light used. Broadspectrum light provided the greatest cell kill following PDT, although LLLT with 635 nm or 905 nm also increased cell kill as compared to PDT alone. All LLLT regimens increased PBR expression compared to controls with corresponding increases in PpIX production. These data suggest that by selectively increasing PBR expression in tumour cells, LLLT may facilitate enhanced cell kill using ALA-PDT without damaging surrounding normal brain.

  11. Widespread ectopic expression of olfactory receptor genes

    PubMed Central

    Feldmesser, Ester; Olender, Tsviya; Khen, Miriam; Yanai, Itai; Ophir, Ron; Lancet, Doron

    2006-01-01

    Background Olfactory receptors (ORs) are the largest gene family in the human genome. Although they are expected to be expressed specifically in olfactory tissues, some ectopic expression has been reported, with special emphasis on sperm and testis. The present study systematically explores the expression patterns of OR genes in a large number of tissues and assesses the potential functional implication of such ectopic expression. Results We analyzed the expression of hundreds of human and mouse OR transcripts, via EST and microarray data, in several dozens of human and mouse tissues. Different tissues had specific, relatively small OR gene subsets which had particularly high expression levels. In testis, average expression was not particularly high, and very few highly expressed genes were found, none corresponding to ORs previously implicated in sperm chemotaxis. Higher expression levels were more common for genes with a non-OR genomic neighbor. Importantly, no correlation in expression levels was detected for human-mouse orthologous pairs. Also, no significant difference in expression levels was seen between intact and pseudogenized ORs, except for the pseudogenes of subfamily 7E which has undergone a human-specific expansion. Conclusion The OR superfamily as a whole, show widespread, locus-dependent and heterogeneous expression, in agreement with a neutral or near neutral evolutionary model for transcription control. These results cannot reject the possibility that small OR subsets might play functional roles in different tissues, however considerable care should be exerted when offering a functional interpretation for ectopic OR expression based only on transcription information. PMID:16716209

  12. Cannabinoid-receptor expression in human leukocytes.

    PubMed

    Bouaboula, M; Rinaldi, M; Carayon, P; Carillon, C; Delpech, B; Shire, D; Le Fur, G; Casellas, P

    1993-05-15

    Marijuana and many of its constituent cannabinoids influence the central nervous system (CNS), probably through the cannabinoid receptor, which has recently been cloned in rat and human. While numerous reports have also described effects of cannabinoids on the immune system, the observation of both mRNA and cannabinoid receptor has hitherto been exclusively confined to the brain, a reported detection in the testis being the sole example of its presence at the periphery. Here we report the expression of the cannabinoid receptor on human immune tissues using a highly sensitive polymerase-chain-reaction-based method for mRNA quantification. We show that, although present in a much lower abundance than in brain, cannabinoid receptor transcripts are found in human spleen, tonsils and peripheral blood leukocytes. The distribution pattern displays important variations of the mRNA level for the cannabinoid receptor among the main human blood cell subpopulations. The rank order of mRNA levels in these cells is B cells > natural killer cells > or = polymorphonuclear neutrophils > or = T8 cells > monocytes > T4 cells. Cannabinoid-receptor mRNA, which is also found in monocytic, as well as T and B leukemia cell lines but not in Jurkat cells, presents a great diversity of expression on these cells as well, B-cell lines expressing a much higher level than T-cell lines. The cannabinoid receptor PCR products from leukocytes and brain are identical both in size and sequence suggesting a strong similarity between central and peripheral cannabinoid receptors. The expression of this receptor was demonstrated on membranes of the myelomonocytic U937 cells using the synthetic cannabinoid [3H]CP-55940 as ligand. The Kd determined from Scatchard analysis was 0.1 nM and the Bmax for membranes was 525 fmol/mg protein. The demonstration of cannabinoid-receptor expression at both mRNA and protein levels on human leukocytes provides a molecular basis for cannabinoid action on these cells. PMID

  13. Developmental changes in the hypothalamic mRNA expression levels of PACAP and its receptor PAC1 and their sensitivity to fasting in male and female rats.

    PubMed

    Iwasa, Takeshi; Matsuzaki, Toshiya; Tungalagsuvd, Altankhuu; Munkhzaya, Munkhsaikhan; Yiliyasi, Maira; Kato, Takeshi; Kuwahara, Akira; Irahara, Minoru

    2016-08-01

    The actions and responses of hypothalamic appetite regulatory and factors change markedly during the neonatal to pre-pubertal period. Pituitary adenylate cyclase-activating polypeptide (PACAP) has been found to play pivotal roles in the regulation of metabolic and nutritional status through its specific receptor PAC1. PACAP/PAC1 have anorectic roles, and their functions are regulated by leptin in adulthood. In the present study, we showed that hypothalamic PACAP mRNA expression decreases during the neonatal to pre-pubertal period (from postnatal day 10-30) in both male and female rats. During this period, hypothalamic PACAP mRNA expression was not affected by 24h fasting in either sex, while the serum leptin levels (leptin is a positive regulator of hypothalamic PACAP expression in adulthood) of both sexes were decreased by fasting. On the other hand, hypothalamic PAC1 mRNA expression did not change during the neonatal to pre-pubertal period in either sex; however, its levels were consistently higher in males than in females. Hypothalamic PAC1 mRNA expression was decreased by 24h fasting in males, but no such changes were observed in females. These results indicate while hypothalamic PACAP expression is sensitive to a negative energy state and the serum leptin level in adulthood, no such relationships are seen in the pre-pubertal period. In addition, we speculate that differences in the gonadal steroidal milieu might induce sexual dimorphism in the basal hypothalamic PAC1 mRNA level and its response to fasting. The mechanisms responsible for and the physiological effects of such changes in hypothalamic PACAP and PAC1 expression during the developmental period remain to be clarified. PMID:27181029

  14. Localized low-level re-expression of high-affinity mesolimbic nicotinic acetylcholine receptors restores nicotine-induced locomotion but not place conditioning.

    PubMed

    Mineur, Y S; Brunzell, D H; Grady, S R; Lindstrom, J M; McIntosh, J M; Marks, M J; King, S L; Picciotto, M R

    2009-04-01

    High-affinity, beta2-subunit-containing (beta2*) nicotinic acetylcholine receptors (nAChRs) are essential for nicotine reinforcement; however, these nAChRs are found on both gamma-aminobutyric acid (GABA) and dopaminergic (DA) neurons in the ventral tegmental area (VTA) and also on terminals of glutamatergic and cholinergic neurons projecting from the pedunculopontine tegmental area and the laterodorsal tegmental nucleus. Thus, systemic nicotine administration stimulates many different neuronal subtypes in various brain nuclei. To identify neurons in which nAChRs must be expressed to mediate effects of systemic nicotine, we investigated responses in mice with low-level, localized expression of beta2* nAChRs in the midbrain/VTA. Nicotine-induced GABA and DA release were partially rescued in striatal synaptosomes from transgenic mice compared with tissue from beta2 knockout mice. Nicotine-induced locomotor activation, but not place preference, was rescued in mice with low-level VTA expression, suggesting that low-level expression of beta2* nAChRs in DA neurons is not sufficient to support nicotine reward. In contrast to control mice, transgenic mice with low-level beta2* nAChR expression in the VTA showed no increase in overall levels of cyclic AMP response element-binding protein (CREB) but did show an increase in CREB phosphorylation in response to exposure to a nicotine-paired chamber. Thus, CREB activation in the absence of regulation of total CREB levels during place preference testing was not sufficient to support nicotine place preference in beta2 trangenic mice. This suggests that partial activation of high-affinity nAChRs in VTA might block the rewarding effects of nicotine, providing a potential mechanism for the ability of nicotinic partial agonists to aid in smoking cessation. PMID:19077117

  15. Amelioration of palmitate-induced metabolic dysfunction in L6 muscle cells expressing low levels of receptor-interacting protein 140.

    PubMed

    Constantinescu, Silvana; Turcotte, Lorraine P

    2015-11-01

    We have shown that reduced expression of receptor-interacting protein 140 (RIP140) alters the regulation of fatty-acid (FA) oxidation in muscle. To determine whether a high level of FA availability alters the effects of RIP140 on metabolic regulation, L6 myotubes were transfected with or without RNA interference oligonucleotide sequences to reduce RIP140 expression, and then incubated with high levels of palmitic acid, with or without insulin. High levels of palmitate reduced basal (53%-58%) and insulin-treated (24%-44%) FA uptake and oxidation, and increased basal glucose uptake (88%). In cells incubated with high levels of palmitate, low RIP140 increased basal FA uptake and insulin-treated FA oxidation and glucose uptake, and decreased basal glucose uptake and insulin-treated FA uptake. Under basal conditions, low RIP140 increased the mRNA content of FAT/CD36 (159%) and COX4 (61%), as well as the protein content of Nur77 (68%), whereas the mRNA expression of FGF21 (50%) was decreased, as was the protein content of CPT1b (35%) and FGF21 (44%). Under insulin-treated conditions, low RIP140 expression increased the mRNA content of MCAD (84%) and Nur77 (84%), as well as the protein content of Nur77 (23%). Thus, a low level of RIP140 restores the rates of FA uptake in the basal state, in part via a reduction in upstream insulin signaling. Our data also indicate that the protein expression of Nur77 may be modulated by RIP140 when muscle cells are metabolically challenged by high levels of palmitate. PMID:26406163

  16. Distinct Transcript Isoforms of the Atypical Chemokine Receptor 1 (ACKR1) / Duffy Antigen Receptor for Chemokines (DARC) Gene Are Expressed in Lymphoblasts and Altered Isoform Levels Are Associated with Genetic Ancestry and the Duffy-Null Allele

    PubMed Central

    Davis, Melissa B.; Walens, Andrea; Hire, Rupali; Mumin, Kauthar; Brown, Andrea M.; Ford, DeJuana; Howerth, Elizabeth W.; Monteil, Michele

    2015-01-01

    The Atypical ChemoKine Receptor 1 (ACKR1) gene, better known as Duffy Antigen Receptor for Chemokines (DARC or Duffy), is responsible for the Duffy Blood Group and plays a major role in regulating the circulating homeostatic levels of pro-inflammatory chemokines. Previous studies have shown that one common variant, the Duffy Null (Fy-) allele that is specific to African Ancestry groups, completely removes expression of the gene on erythrocytes; however, these individuals retain endothelial expression. Additional alleles are associated with a myriad of clinical outcomes related to immune responses and inflammation. In addition to allele variants, there are two distinct transcript isoforms of DARC which are expressed from separate promoters, and very little is known about the distinct transcriptional regulation or the distinct functionality of these protein isoforms. Our objective was to determine if the African specific Fy- allele alters the expression pattern of DARC isoforms and therefore could potentially result in a unique signature of the gene products, commonly referred to as antigens. Our work is the first to establish that there is expression of DARC on lymphoblasts. Our data indicates that people of African ancestry have distinct relative levels of DARC isoforms expressed in these cells. We conclude that the expression of both isoforms in combination with alternate alleles yields multiple Duffy antigens in ancestry groups, depending upon the haplotypes across the gene. Importantly, we hypothesize that DARC isoform expression patterns will translate into ancestry-specific inflammatory responses that are correlated with the axis of pro-inflammatory chemokine levels and distinct isoform-specific interactions with these chemokines. Ultimately, this work will increase knowledge of biological mechanisms underlying disparate clinical outcomes of inflammatory-related diseases among ethnic and geographic ancestry groups. PMID:26473357

  17. Disruption of Aryl Hydrocarbon Receptor Homeostatic Levels during Embryonic Stem Cell Differentiation Alters Expression of Homeobox Transcription Factors that Control Cardiomyogenesis

    PubMed Central

    Wang, Qin; Chen, Jing; Ko, Chia-I; Fan, Yunxia; Carreira, Vinicius; Chen, Yinglei; Xia, Ying; Medvedovic, Mario

    2013-01-01

    Background: The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that regulates the expression of xenobiotic detoxification genes and is a critical mediator of gene–environment interactions. Many AHR target genes identified by genome-wide gene expression profiling have morphogenetic functions, suggesting that AHR may play a role in embryonic development. Objectives: To characterize the developmental functions of the AHR, we studied the consequences of AHR activation by the agonist 2,3,7,8-tetrachlorodibenzo-p-doxin (TCDD), and the result of its repression by the antagonists 6,2,4-trimethoxyflavone and CH 223191 or by short-hairpin RNA (shRNA)-mediated Ahr knockdown during spontaneous differentiation of embryonic stem (ES) cells into cardiomyocytes. Methods: We generated an AHR-positive cardiomyocyte lineage differentiated from mouse ES cells that expresses puromycin resistance and enhanced green fluorescent protein (eGFP) under the control of the Cyp1a1 (cytochrome P450 1a1) promoter. We used RNA sequencing (RNA.Seq) to analyze temporal trajectories of TCDD-dependent global gene expression in these cells during differentiation. Results: Activation, inhibition, and knockdown of Ahr significantly inhibited the formation of contractile cardiomyocyte nodes. Global expression analysis of AHR-positive cells showed that activation of the AHR/TCDD axis disrupted the concerted expression of genes that regulate multiple signaling pathways involved in cardiac and neural morphogenesis and differentiation, including dozens of genes encoding homeobox transcription factors and Polycomb and trithorax group proteins. Conclusions: Disruption of AHR expression levels resulted in gene expression changes that perturbed cardiomyocyte differentiation. The main function of the AHR during development appears to be the coordination of a complex regulatory network responsible for attainment and maintenance of cardiovascular homeostasis. Citation: Wang Q, Chen J

  18. Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells.

    PubMed

    Mina, Marco; Magi, Shigeyuki; Jurman, Giuseppe; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R R; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O; Okada-Hatakeyama, Mariko; Furlanello, Cesare

    2015-01-01

    The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors. PMID:26179713

  19. Promoter-level expression clustering identifies time development of transcriptional regulatory cascades initiated by ErbB receptors in breast cancer cells

    PubMed Central

    Mina, Marco; Magi, Shigeyuki; Jurman, Giuseppe; Itoh, Masayoshi; Kawaji, Hideya; Lassmann, Timo; Arner, Erik; Forrest, Alistair R. R.; Carninci, Piero; Hayashizaki, Yoshihide; Daub, Carsten O.; Okada-Hatakeyama, Mariko; Furlanello, Cesare

    2015-01-01

    The analysis of CAGE (Cap Analysis of Gene Expression) time-course has been proposed by the FANTOM5 Consortium to extend the understanding of the sequence of events facilitating cell state transition at the level of promoter regulation. To identify the most prominent transcriptional regulations induced by growth factors in human breast cancer, we apply here the Complexity Invariant Dynamic Time Warping motif EnRichment (CIDER) analysis approach to the CAGE time-course datasets of MCF-7 cells stimulated by epidermal growth factor (EGF) or heregulin (HRG). We identify a multi-level cascade of regulations rooted by the Serum Response Factor (SRF) transcription factor, connecting the MAPK-mediated transduction of the HRG stimulus to the negative regulation of the MAPK pathway by the members of the DUSP family phosphatases. The finding confirms the known primary role of FOS and FOSL1, members of AP-1 family, in shaping gene expression in response to HRG induction. Moreover, we identify a new potential regulation of DUSP5 and RARA (known to antagonize the transcriptional regulation induced by the estrogen receptors) by the activity of the AP-1 complex, specific to HRG response. The results indicate that a divergence in AP-1 regulation determines cellular changes of breast cancer cells stimulated by ErbB receptors. PMID:26179713

  20. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor-binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Y.; Robinson, H.; Gao, X.; Qin, L.; Buchko, G. W.; Varnum, S. M.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.

  1. High-level Expression Purification Crystallization and Preliminary X-ray Crystallographic Studies of the Receptor Binding Domain of botulinum neurotoxin Serotype D

    SciTech Connect

    Y Zhang; X Gao; G Buchko; H Robinson; S Varnum

    2011-12-31

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and animals that are responsible for the deadly neuroparalytic disease botulism. Here, details of the expression and purification of the receptor-binding domain (HCR) of BoNT/D in Escherichia coli are presented. Using a codon-optimized cDNA, BoNT/D{_}HCR was expressed at a high level (150-200 mg per litre of culture) in the soluble fraction. Following a three-step purification protocol, very pure (>98%) BoNT/D{_}HCR was obtained. The recombinant BoNT/D{_}HCR was crystallized and the crystals diffracted to 1.65 {angstrom} resolution. The crystals belonged to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 60.8, b = 89.7, c = 93.9 {angstrom}. Preliminary crystallographic data analysis revealed the presence of one molecule in the asymmetric unit.

  2. High-level expression, purification, crystallization and preliminary X-ray crystallographic studies of the receptor binding domain of botulinum neurotoxin serotype D

    SciTech Connect

    Zhang, Yanfeng; Gao, Xiaoli; Qin, Lin; Buchko, Garry W.; Robinson, Howard; Varnum, Susan M.

    2010-12-01

    Botulinum neurotoxins (BoNTs) are highly toxic proteins for humans and can cause neuroparalytic disease botulism. Due to the limitations of production and manipulation of holoenzymes, expressing non-toxic heavy chain receptor binding domains (HCR) has become a common strategy for vaccine and antibody development. Meanwhile, large quantities and highly purified soluble proteins are required for research areas such as antibody maturation and structural biology. We present high level expression and purification of the BoNT serotype D HCR in E. coli using a codon-optimized cDNA. By varying expression conditions, especially at low temperature, the protein was expressed at a high level with high solubility. About 150-200 mg protein was purified to >90% purity from 1 L cell culture. The recombinant D_HCR was crystallized and the crystals diffracted to 1.65 Å resolution. The crystals belong to space group P212121 with unit cell dimensions a = 60.8 Å, b = 89.7 Å, c = 93.9 Å. Preliminary crystallographic data analysis revealed one molecule in asymmetric unit.

  3. Leptin receptor null mice with reexpression of LepR in GnRHR expressing cells display elevated FSH levels but remain in a prepubertal state.

    PubMed

    Allen, Susan J; Garcia-Galiano, David; Borges, Beatriz C; Burger, Laura L; Boehm, Ulrich; Elias, Carol F

    2016-06-01

    Leptin signals energy sufficiency to the reproductive hypothalamic-pituitary-gonadal (HPG) axis. Studies using genetic models have demonstrated that hypothalamic neurons are major players mediating these effects. Leptin receptor (LepR) is also expressed in the pituitary gland and in the gonads, but the physiological effects of leptin in these sites are still unclear. Female mice with selective deletion of LepR in a subset of gonadotropes show normal pubertal development but impaired fertility. Conditional deletion approaches, however, often result in redundancy or developmental adaptations, which may compromise the assessment of leptin's action in gonadotropes for pubertal maturation. To circumvent these issues, we adopted a complementary genetic approach and assessed if selective reexpression of LepR only in gonadotropes is sufficient to enable puberty and improve fertility of LepR null female mice. We initially assessed the colocalization of gonadotropin-releasing hormone receptor (GnRHR) and LepR in the HPG axis using GnRHR-IRES-Cre (GRIC) and LepR-Cre reporter (tdTomato or enhanced green fluorescent protein) mice. We found that GRIC and leptin-induced phosphorylation of STAT3 are expressed in distinct hypothalamic neurons. Whereas LepR-Cre was observed in theca cells, GRIC expression was rarely found in the ovarian parenchyma. In contrast, a subpopulation of gonadotropes expressed the LepR-Cre reporter gene (tdTomato). We then crossed the GRIC mice with the LepR null reactivable (LepR(loxTB)) mice. These mice showed an increase in FSH levels, but they remained in a prepubertal state. Together with previous findings, our data indicate that leptin-selective action in gonadotropes serves a role in adult reproductive physiology but is not sufficient to allow pubertal maturation in mice. PMID:27101301

  4. Raloxifene induces cell death and inhibits proliferation through multiple signaling pathways in prostate cancer cells expressing different levels of estrogen receptor α and β.

    PubMed

    Rossi, V; Bellastella, G; De Rosa, C; Abbondanza, C; Visconti, D; Maione, L; Chieffi, P; Della Ragione, F; Prezioso, D; De Bellis, A; Bellastella, A; Sinisi, A A

    2011-05-01

    Raloxifene (RAL), a selective estrogen receptor (ER) modulator (SERM) seems to induce apoptosis in both androgen-dependent and -independent prostate cell (PC) lines via activation of ERβ and an antagonistic effect on ERα. In this study, we evaluated the effects of RAL on epithelial PC growth using the two following in vitro models: the androgen-dependent cell line EPN which expressed both ERs; and a stabilized epithelial cell line derived from a prostate cancer specimen (CPEC), which expressed low levels of ERβ and lacked ERα. In EPN cells, there was an increase in the pre-G1 apoptotic peak and a reduction in the S phase of the cell cycle with G0/G1 arrest after E2 or RAL treatment; bcl-2 mRNA and Bcl-2 protein levels were significantly reduced, while activated caspase-3 and Par-4 levels increased significantly after either E2 or RAL treatment; in addition, c-myc transcript was inhibited after 10(-6)  M RAL treatment. A dose-dependent increase of metallothionein II gene RNA level was also induced by RAL in EPN. In CPEC, there was only a weak apoptotic peak associated with caspase-3 activation and Par-4 increase after either E2 or RAL treatment; while c-myc transcript level increased. RAL induced a rapid but transient phosphorylation of ERK 1/2 in EPN cells but generated a sustained effect in CPEC. These findings suggest that RAL effects on PC growth control in vitro are cell-specific, depending on ERβ or ERβ/ERα relative expression levels. Moreover, this study demonstrated that RAL affected both transcriptional regulation and non-genomic signals, which resulted in the modulation of multiple signaling pathways of apoptosis and of cell cycle progression. PMID:20945400

  5. Effect of fluoride on insulin level of rats and insulin receptor expression in the MC3T3-E1 cells.

    PubMed

    Hu, Chun-Yan; Ren, Li-Qun; Li, Xi-Ning; Wu, Nan; Li, Guang-Sheng; Liu, Qin-Yi; Xu, Hui

    2012-12-01

    Studies on the role of insulin and insulin receptor (InsR) in the process of skeletal fluorosis, especially in osteogenic function, are rare. We evaluated the effect of increasing F⁻ doses on the marker of bone formation, serum insulin level and pancreatic secretion changes in vivo and mRNA expression of InsR and osteocalcin (OCN) in vitro. Wistar rats (n = 50) were divided into two groups, i.e. a control group and fluoride group. The fluoride groups were treated with fluoride by drinking tap water containing 100 mg F⁻/L. The fluoride ion-selective electrode measured the fluoride concentrations of femurs. The alkaline phosphatase (ALP), OCN, insulin and glucagon of serum were tested to observe the effect of fluoride action on them. Meantime, the pancreas pathological morphometry analysis via β cells stained by aldehyde fuchsin showed the action of fluoride on pancreas secretion. MC3T3-E1 cells (derived from newborn mouse calvaria) were exposed to varying concentrations and periods of fluoride. The mRNA expression of InsR and OCN was quantified with real-time PCR. Results showed that 1-year fluoride treatment obviously stimulated ALP activity and OCN level along with increase of bone fluoride concentration of rats, which indicated that fluoride obviously stimulated osteogenic action of rats. In vitro study, the dual effect of fluoride on osteoblast function is shown. On the other hand, there was a significant increase of serum insulin level and a general decrease of glucagon level, and the histomorphometry analysis indicated an elevated insulin-positive area and increase in islet size in rats treated with fluoride for 1 year. In addition, fluoride obviously facilitated the mRNA expression of InsR in vitro. To sum up, there existed a close relationship between insulin secretion and fluoride treatment. The insulin signal pathway might be involved in the underlying occurrence or development of skeletal fluorosis. PMID:22872571

  6. Expression and function of ryanodine receptors in nonexcitable cells.

    PubMed

    Bennett, D L; Cheek, T R; Berridge, M J; De Smedt, H; Parys, J B; Missiaen, L; Bootman, M D

    1996-03-15

    We have used reverse transcriptase-polymerase chain reaction to investigate the expression of ryanodine receptors in several excitable and nonexcitable cell types. Consistent with previous reports, we detected ryanodine receptor expression in brain, heart, and skeletal muscle. In addition, we detected ryanodine receptor expression in various other excitable cells including PC 12 and A7r5 cells. Several muscle cell lines (BC3H1, C2C12, L6, and Sol8) weakly expressed ryanodine receptor when undifferentiated but strongly expressed type 1 and type 3 ryanodine receptor isoforms when differentiated into a muscle phenotype. Only 2 (HeLa and LLC-PK1 cells) out of 11 nonexcitable cell types examined expressed ryanodine receptors. Expression of ryanodine receptors at the protein level in these cells was confirmed using [3H]ryanodine binding. We also investigated the function of ryanodine receptors in Ca2+ signaling in HeLa cells using single-cell Fura-2 imaging. Neither caffeine nor ryanodine caused a detectable elevation of cytoplasmic Ca2+ in single HeLa cells. However, ryanodine caused a significant decrease in the amplitude of Ca 2+ signals evoked by repetitive stimulation with ATP. These studies show that ryanodine receptors are expressed in some nonexcitable cell types and furthermore suggest that the ryanodine receptors may be involved in a subtle regulation of intracellular Ca2+ responses. PMID:8626432

  7. A link between high serum levels of human chorionic gonadotrophin and chorionic expression of its mature functional receptor (LHCGR) in Down's syndrome pregnancies

    PubMed Central

    Banerjee, Subhasis; Smallwood, Alan; Chambers, Anne E; Papageorghiou, Aris; Loosfelt, Hugues; Spencer, Kevin; Campbell, Stuart; Nicolaides, Kypros

    2005-01-01

    Human chorionic gonadotrophin (hCG) is released from placental trophoblasts and is involved in establishing pregnancy by maintaining progesterone secretion from the corpus luteum. Serum hCG is detected in the maternal circulation within the first 2–3 wks of gestation and peaks at the end of the first trimester before declining. In Down's syndrome (DS) pregnancies, serum hCG remains significantly high compared to gestation age-matched uncompromised pregnancies. It has been proposed that increased serum hCG levels could be due to transcriptional hyper-activation of the CGB (hCG beta) gene, or an increased half life of glycosylated hCG hormone, or both. Another possibility is that serum hCG levels remain high due to reduced availability of the hormone's cognate receptor, LHCGR, leading to lack of hormone utilization. We have tested this hypothesis by quantifying the expression of the hCG beta (CGB) RNA, LHCGR RNA and LHCGR proteins in chorionic villous samples. We demonstrate that chorionic expression of hCG beta (CGB) mRNA directly correlates with high serum hCG levels. The steady-state synthesis of LHCGR mRNA (exons 1–5) in DS pregnancies was significantly higher than that of controls, but the expression of full-length LHCGR mRNA (exons 1–11) in DS was comparable to that of uncompromised pregnancies. However, the synthesis of high molecular weight mature LHCGR proteins was significantly reduced in DS compared to uncompromised pregnancies, suggesting a lack of utilization of circulating hCG in DS pregnancies. PMID:15969756

  8. Effects of Starvation on Brain Short Neuropeptide F-1, -2, and -3 Levels and Short Neuropeptide F Receptor Expression Levels of the Silkworm, Bombyx mori

    PubMed Central

    Nagata, Shinji; Matsumoto, Sumihiro; Nakane, Tomohiro; Ohara, Ayako; Morooka, Nobukatsu; Konuma, Takahiro; Nagai, Chiaki; Nagasawa, Hiromichi

    2012-01-01

    In our previous report, we demonstrated the possibility that various regulatory neuropeptides influence feeding behavior in the silkworm, Bombyx mori. Among these feeding-related neuropeptides, short neuropeptide F (sNPF) exhibited feeding-accelerating activity when injected into B. mori larvae. Like other insect sNPFs, the deduced amino acid sequence of the cDNA encoding the sNPF precursor appears to produce multiple sNPF and sNPF-related peptides in B. mori. The presence of three sNPFs, sNPF-1, sNPF-2, and sNPF-3, in the brain of B. mori larvae was confirmed by direct MALDI-TOF mass spectrometric profiling. In addition, all three sNPFs are present in other larval ganglia. The presence of sNPF mRNA in the central nervous system (CNS) was also confirmed by Reverse transcription-polymerase chain reaction. Semi-quantitative analyses of sNPFs in the larval brain using matrix-assisted laser desorption ionization time-of-flight mass spectrometry further revealed that brain sNPF levels decrease in response to starvation, and that they recover with the resumption of feeding. These data suggest that sNPFs were depleted by the starvation process. Furthermore, food deprivation decreased the transcriptional levels of the sNPF receptor (BNGR-A10) in the brain and CNS, suggesting that the sNPF system is dependent on the feeding state of the insect and that the sNPF system may be linked to locomotor activity associated with foraging behavior. Since the injection of sNPFs accelerated the onset of feeding in B. mori larvae, we concluded that sNPFs are strongly related to feeding behavior. In addition, semi-quantitative MS analyses revealed that allatostatin, which is present in the larval brain, is also reduced in response to starvation, whereas the peptide level of Bommyosuppressin was not affected by different feeding states. PMID:22649403

  9. IL-21 Receptor Expression in Human Tendinopathy

    PubMed Central

    Campbell, Abigail L.; Smith, Nicola C.; Reilly, James H.; Kerr, Shauna C.; Leach, William J.; Fazzi, Umberto G.; Rooney, Brian P.; Murrell, George A. C.; Millar, Neal L.

    2014-01-01

    The pathogenetic mechanisms underlying tendinopathy remain unclear, with much debate as to whether inflammation or degradation has the prominent role. Increasing evidence points toward an early inflammatory infiltrate and associated inflammatory cytokine production in human and animal models of tendon disease. The IL-21/IL-21R axis is a proinflammatory cytokine complex that has been associated with chronic inflammatory diseases including rheumatoid arthritis and inflammatory bowel disease. This project aimed to investigate the role and expression of the cytokine/receptor pair IL-21/IL-21R in human tendinopathy. We found significantly elevated expression of IL-21 receptor message and protein in human tendon samples but found no convincing evidence of the presence of IL-21 at message or protein level. The level of expression of IL-21R message/protein in human tenocytes was significantly upregulated by proinflammatory cytokines (TNFα/IL-1β) in vitro. These findings demonstrate that IL-21R is present in early human tendinopathy mainly expressed by tenocytes and macrophages. Despite a lack of IL-21 expression, these data again suggest that early tendinopathy has an inflammatory/cytokine phenotype, which may provide novel translational targets in the treatment of tendinopathy. PMID:24757284

  10. 5-HT1A receptor gene silencers Freud-1 and Freud-2 are differently expressed in the brain of rats with genetically determined high level of fear-induced aggression or its absence.

    PubMed

    Kondaurova, Elena M; Ilchibaeva, Tatiana V; Tsybko, Anton S; Kozhemyakina, Rimma V; Popova, Nina K; Naumenko, Vladimir S

    2016-09-01

    Serotonin 5-HT1A receptor is known to play a crucial role in the mechanisms of genetically defined aggression. In its turn, 5-HT1A receptor functional state is under control of multiple factors. Among others, transcriptional factors Freud-1 and Freud-2 are known to be involved in the repression of 5-HT1A receptor gene expression. However, implication of these factors in the regulation of behavior is unclear. Here, we investigated the expression of 5-HT1A receptor and silencers Freud-1 and Freud-2 in the brain of rats selectively bred for 85 generations for either high level of fear-induced aggression or its absence. It was shown that Freud-1 and Freud-2 levels were different in aggressive and nonaggressive animals. Freud-1 protein level was decreased in the hippocampus, whereas Freud-2 protein level was increased in the frontal cortex of highly aggressive rats. There no differences in 5-HT1A receptor gene expression were found in the brains of highly aggressive and nonaggressive rats. However, 5-HT1A receptor protein level was decreased in the midbrain and increased in the hippocampus of highly aggressive rats. These data showed the involvement of Freud-1 and Freud-2 in the regulation of genetically defined fear-induced aggression. However, these silencers do not affect transcription of the 5-HT1A receptor gene in the investigated rats. Our data indicate the implication of posttranscriptional rather than transcriptional regulation of 5-HT1A receptor functional state in the mechanisms of genetically determined aggressive behavior. On the other hand, the implication of other transcriptional regulators for 5-HT1A receptor gene in the mechanisms of genetically defined aggression could be suggested. PMID:27150226

  11. Increased Expression of Kindlin 2 in Luteinized Granulosa Cells Correlates With Androgen Receptor Level in Patients With Polycystic Ovary Syndrome Having Hyperandrogenemia

    PubMed Central

    Yang, Mei; Du, Juan; Lu, Danyu; Ren, Caixia; Shen, Huan; Qiao, Jie; Zhang, Hongquan

    2014-01-01

    Hyperandrogenemia is the leading defect in patients with polycystic ovary syndrome (PCOS) and considered to be involved in the ovulation dysfunction of PCOS. During the process of ovulation, granulosa cells (GCs) undergo epithelial–mesenchymal transition (EMT), and integrin-interacting protein kindlin 2 is a well-known regulator in EMT. Therefore, our objective here was to compare the expression levels of kindlin 2 in luteinized GCs between patients with PCOS and control women and the relationship between kindlin 2 and PCOS pathogenesis. In this study, kindlin 2 expression was significantly increased in luteinized GCs from patients with PCOS, and kindlin 2 could be induced by testosterone both in vitro and in vivo. Meanwhile, kindlin 2 was positively correlated with androgen receptor (AR) in PCOS GCs. Taken together, kindlin 2 may play a role in luteinized GCs, especially in the case of excess androgen. Further studies are required to assess the specific role of kindlin 2 in follicular development and PCOS pathogenesis. PMID:24336678

  12. N-3 Polyunsaturated Fatty Acids Improve Liver Lipid Oxidation-Related Enzyme Levels and Increased the Peroxisome Proliferator-Activated Receptor α Expression Level in Mice Subjected to Hemorrhagic Shock/Resuscitation

    PubMed Central

    Zhang, Li; Tian, Feng; Gao, Xuejin; Wang, Xinying; Wu, Chao; Li, Ning; Li, Jieshou

    2016-01-01

    Appropriate metabolic interventions after hemorrhagic shock/resuscitation injury have not yet been identified. We aimed to examine the effects of fish oil on lipid metabolic intervention after hemorrhagic shock/resuscitation. Firstly, 48 C57BL/6 mice were assigned to six groups (n = 8 per group). The sham group did not undergo surgery, while mice in the remaining groups were sacrificed 1–5 days after hemorrhagic shock/resuscitation. In the second part, mice were treated with saline or fish oil (n = 8 per group) five days after injury. We determined serum triglyceride levels and liver tissues were collected and prepared for qRT-PCR or Western blot analysis. We found that triglyceride levels were increased five days after hemorrhagic shock/resuscitation, but decreased after addition of fish oil. After injury, the protein and gene expression of carnitine palmitoyltransferase 1A, fatty acid transport protein 1, and peroxisome proliferator-activated receptor-α decreased significantly in liver tissue. In contrast, after treatment with fish oil, the expression levels of these targets increased compared with those in the saline group. The present results suggest n-3 polyunsaturated fatty acids could improve lipid oxidation-related enzymes in liver subjected to hemorrhagic shock/resuscitation. This function is possibly accomplished through activating the peroxisome proliferator-activated receptor-α pathway. PMID:27110821

  13. N-3 Polyunsaturated Fatty Acids Improve Liver Lipid Oxidation-Related Enzyme Levels and Increased the Peroxisome Proliferator-Activated Receptor α Expression Level in Mice Subjected to Hemorrhagic Shock/Resuscitation.

    PubMed

    Zhang, Li; Tian, Feng; Gao, Xuejin; Wang, Xinying; Wu, Chao; Li, Ning; Li, Jieshou

    2016-01-01

    Appropriate metabolic interventions after hemorrhagic shock/resuscitation injury have not yet been identified. We aimed to examine the effects of fish oil on lipid metabolic intervention after hemorrhagic shock/resuscitation. Firstly, 48 C57BL/6 mice were assigned to six groups (n = 8 per group). The sham group did not undergo surgery, while mice in the remaining groups were sacrificed 1-5 days after hemorrhagic shock/resuscitation. In the second part, mice were treated with saline or fish oil (n = 8 per group) five days after injury. We determined serum triglyceride levels and liver tissues were collected and prepared for qRT-PCR or Western blot analysis. We found that triglyceride levels were increased five days after hemorrhagic shock/resuscitation, but decreased after addition of fish oil. After injury, the protein and gene expression of carnitine palmitoyltransferase 1A, fatty acid transport protein 1, and peroxisome proliferator-activated receptor-α decreased significantly in liver tissue. In contrast, after treatment with fish oil, the expression levels of these targets increased compared with those in the saline group. The present results suggest n-3 polyunsaturated fatty acids could improve lipid oxidation-related enzymes in liver subjected to hemorrhagic shock/resuscitation. This function is possibly accomplished through activating the peroxisome proliferator-activated receptor-α pathway. PMID:27110821

  14. Age-Dependent Modifications of AMPA Receptor Subunit Expression Levels and Related Cognitive Effects in 3xTg-AD Mice.

    PubMed

    Cantanelli, Pamela; Sperduti, Samantha; Ciavardelli, Domenico; Stuppia, Liborio; Gatta, Valentina; Sensi, Stefano Luca

    2014-01-01

    GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca(2+)-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q-R substitution, a key factor in the regulation of AMPAR Ca(2+)-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca(2+) and Zn(2+). The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer's disease (AD). With quantitative real-time PCR analysis, we assayed hippocampal mRNA expression levels of GluA1-4 subunits occurring in young [3 months of age (m.o.a.)] and old (12 m.o.a) Tg-AD mice and made comparisons with levels found in age-matched wild type (WT) mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for learning short- and long-term spatial memory with the Morris Water Maze (MWM) navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1-4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in

  15. Age-Dependent Modifications of AMPA Receptor Subunit Expression Levels and Related Cognitive Effects in 3xTg-AD Mice

    PubMed Central

    Cantanelli, Pamela; Sperduti, Samantha; Ciavardelli, Domenico; Stuppia, Liborio; Gatta, Valentina; Sensi, Stefano Luca

    2014-01-01

    GluA1, GluA2, GluA3, and GluA4 are the constitutive subunits of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs), the major mediators of fast excitatory transmission in the mammalian central nervous system. Most AMPARs are Ca2+-impermeable because of the presence of the GluA2 subunit. GluA2 mRNA undergoes an editing process that results in a Q–R substitution, a key factor in the regulation of AMPAR Ca2+-permeability. AMPARs lacking GluA2 or containing the unedited subunit are permeable to Ca2+ and Zn2+. The phenomenon physiologically modulates synaptic plasticity while, in pathologic conditions, leads to increased vulnerability to excitotoxic neuronal death. Given the importance of these subunits, we have therefore evaluated possible associations between changes in expression levels of AMPAR subunits and development of cognitive deficits in 3xTg-AD mice, a widely investigated transgenic mouse model of Alzheimer’s disease (AD). With quantitative real-time PCR analysis, we assayed hippocampal mRNA expression levels of GluA1–4 subunits occurring in young [3 months of age (m.o.a.)] and old (12 m.o.a) Tg-AD mice and made comparisons with levels found in age-matched wild type (WT) mice. Efficiency of GluA2 RNA editing was also analyzed. All animals were cognitively tested for learning short- and long-term spatial memory with the Morris Water Maze (MWM) navigation task. 3xTg-AD mice showed age-dependent decreases of mRNA levels for all the AMPAR subunits, with the exception of GluA2. Editing remained fully efficient with aging in 3xTg-AD and WT mice. A one-to-one correlation analysis between MWM performances and GluA1–4 mRNA expression profiles showed negative correlations between GluA2 levels and MWM performances in young 3xTg-AD mice. On the contrary, positive correlations between GluA2 mRNA and MWM performances were found in young WT mice. Our data suggest that increases of AMPARs that contain GluA1, GluA3, and GluA4 subunits may help in

  16. The alpaca melanocortin 1 receptor: gene mutations, transcripts, and relative levels of expression in ventral skin biopsies.

    PubMed

    Chandramohan, Bathrachalam; Renieri, Carlo; La Manna, Vincenzo; La Terza, Antonietta

    2015-01-01

    The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5'-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3'UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation. PMID:25685836

  17. The Alpaca Melanocortin 1 Receptor: Gene Mutations, Transcripts, and Relative Levels of Expression in Ventral Skin Biopsies

    PubMed Central

    Renieri, Carlo; La Terza, Antonietta

    2015-01-01

    The objectives of the present study were to characterize the MC1R gene, its transcripts and the single nucleotide polymorphisms (SNPs) associated with coat color in alpaca. Full length cDNA amplification revealed the presence of two transcripts, named as F1 and F2, differing only in the length of their 5′-terminal untranslated region (UTR) sequences and presenting a color specific expression. Whereas the F1 transcript was common to white and colored (black and brown) alpaca phenotypes, the shorter F2 transcript was specific to white alpaca. Further sequencing of the MC1R gene in white and colored alpaca identified a total of twelve SNPs; among those nine (four silent mutations (c.126C>A, c.354T>C, c.618G>A, and c.933G>A); five missense mutations (c.82A>G, c.92C>T, c.259A>G, c.376A>G, and c.901C>T)) were observed in coding region and three in the 3′UTR. A 4 bp deletion (c.224 227del) was also identified in the coding region. Molecular segregation analysis uncovered that the combinatory mutations in the MC1R locus could cause eumelanin and pheomelanin synthesis in alpaca. Overall, our data refine what is known about the MC1R gene and provides additional information on its role in alpaca pigmentation. PMID:25685836

  18. Profiling Eph receptor expression in cells and tissues

    PubMed Central

    Noberini, Roberta; Rubio de la Torre, Elena; Pasquale, Elena B.

    2012-01-01

    The Eph receptor tyrosine kinase family includes many members, which are often expressed together in various combinations and can promiscuously interact with multiple ephrin ligands, generating intricate networks of intracellular signals that control physiological and pathological processes. Knowing the entire repertoire of Eph receptors and ephrins expressed in a biological sample is important when studying their biological roles. Moreover, given the correlation between Eph receptor/ephrin expression and cancer pathogenesis, their expression patterns could serve important diagnostic and prognostic purposes. However, profiling Eph receptor and ephrin expression has been challenging. Here we describe a novel and straightforward approach to catalog the Eph receptors present in cultured cells and tissues. By measuring the binding of ephrin Fc fusion proteins to Eph receptors in ELISA and pull-down assays, we determined that a mixture of four ephrins is suitable for isolating both EphA and EphB receptors in a single pull-down. We then used mass spectrometry to identify the Eph receptors present in the pull-downs and estimate their relative levels. This approach was validated in cultured human cancer cell lines, human tumor xenograft tissue grown in mice, and mouse brain tissue. The new mass spectrometry approach we have developed represents a useful tool for the identification of the spectrum of Eph receptors present in a biological sample and could also be extended to profiling ephrin expression. PMID:22568954

  19. Hormone activation of baculovirus expressed progesterone receptors.

    PubMed

    Elliston, J F; Beekman, J M; Tsai, S Y; O'Malley, B W; Tsai, M J

    1992-03-15

    Human and chicken progesterone receptors (A form) were overproduced in a baculovirus expression system. These recombinant progesterone receptors were full-length bound progesterone specifically and were recognized by monoclonal antibodies, AB52 and PR22, specific for human and chicken progesterone receptor, respectively. In gel retardation studies, binding of recombinant human and chicken progesterone receptors to their progesterone response element (PRE) was specific and was enhanced in the presence of progesterone. Binding of human progesterone receptor to the PRE was also enhanced in the presence of the antiprogestin, RU486, but very little effect was observed in the presence of estradiol, dexamethasone, testosterone, and vitamin D. In our cell-free transcription system, human progesterone receptor induced transcription in a receptor-dependent and hormone-activable manner. Receptor-stimulated transcription required the presence of the PRE in the test template and could be specifically inhibited by excess PRE oligonucleotides. Furthermore, chicken progesterone receptor also induced in vitro transcription in a hormone-activable manner. These results demonstrate that steroid receptors overexpressed in a baculovirus expression system are functional and exhibit steroid-responsive binding and transcription. These observations support our present understanding of the mechanism of steroid receptor-regulated gene expression and provide a technological format for studies of the role of hormone and antihormone in altering gene expression. PMID:1544902

  20. Human neuroepithelial cells express NMDA receptors.

    PubMed

    Sharp, Christopher D; Fowler, M; Jackson, T H; Houghton, J; Warren, A; Nanda, A; Chandler, I; Cappell, B; Long, A; Minagar, A; Alexander, J S

    2003-11-13

    L-glutamate, an excitatory neurotransmitter, binds to both ionotropic and metabotropic glutamate receptors. In certain parts of the brain the BBB contains two normally impermeable barriers: 1) cerebral endothelial barrier and 2) cerebral epithelial barrier. Human cerebral endothelial cells express NMDA receptors; however, to date, human cerebral epithelial cells (neuroepithelial cells) have not been shown to express NMDA receptor message or protein. In this study, human hypothalamic sections were examined for NMDA receptors (NMDAR) expression via immunohistochemistry and murine neuroepithelial cell line (V1) were examined for NMDAR via RT-PCR and Western analysis. We found that human cerebral epithelium express protein and cultured mouse neuroepithelial cells express both mRNA and protein for the NMDA receptor. These findings may have important consequences for neuroepithelial responses during excitotoxicity and in disease. PMID:14614784

  1. Transient neuromotor phenotype in transgenic spastic mice expressing low levels of glycine receptor β-subunit: an animal model of startle disease

    PubMed Central

    Becker, Lore; Hartenstein, Bettina; Schenkel, Johannes; Kuhse, Jochen; Betz, Heinrich; Weiher, Hans

    2000-01-01

    Startle disease or hereditary hyperekplexia has been shown to result from mutations in the α1-subunit gene of the inhibitory glycine receptor (GlyR). In hyperekplexia patients, neuromotor symptoms generally become apparent at birth, improve with age, and often disappear in adulthood. Loss-of-function mutations of GlyR α or β-subunits in mice show rather severe neuromotor phenotypes. Here, we generated mutant mice with a transient neuromotor deficiency by introducing a GlyR β transgene into the spastic mouse (spa/spa), a recessive mutant carrying a transposon insertion within the GlyR β-subunit gene. In spa/spa TG456 mice, one of three strains generated with this construct, which expressed very low levels of GlyR β transgene-dependent mRNA and protein, the spastic phenotype was found to depend upon the transgene copy number. Notably, mice carrying two copies of the transgene showed an age-dependent sensitivity to tremor induction, which peaked at ∼ 3–4 weeks postnatally. This closely resembles the development of symptoms in human hyperekplexia patients, where motor coordination significantly improves after adolescence. The spa/spa TG456 line thus may serve as an animal model of human startle disease. PMID:10651857

  2. Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

    PubMed

    Imbesi, M; Yildiz, S; Dirim Arslan, A; Sharma, R; Manev, H; Uz, T

    2009-01-23

    Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  3. Hypothyroidism affects D2 receptor-mediated breathing without altering D2 receptor expression.

    PubMed

    Schlenker, Evelyn H; Del Rio, Rodrigo; Schultz, Harold D

    2014-03-01

    Bromocriptine depressed ventilation in air and D2 receptor expression in the nucleus tractus solitaries (NTS) in male hypothyroid hamsters. Here we postulated that in age-matched hypothyroid female hamsters, the pattern of D2 receptor modulation of breathing and D2 receptor expression would differ from those reported in hypothyroid males. In females hypothyroidism did not affect D2 receptor protein levels in the NTS, carotid bodies or striatum. Bromocriptine, but not carmoxirole (a peripheral D2 receptor agonist), increased oxygen consumption and body temperature in awake air-exposed hypothyroid female hamsters and stimulated their ventilation before and following exposure to hypoxia. Carmoxirole depressed frequency of breathing in euthyroid hamsters prior to, during and following hypoxia exposures and stimulated it in the hypothyroid hamsters following hypoxia. Although hypothyroidism did not affect expression of D2 receptors, it influenced central D2 modulation of breathing in a disparate manner relative to euthyroid hamsters. PMID:24434437

  4. Alteration at translational but not transcriptional level of transferrin receptor expression following manganese exposure at the blood-CSF barrier in vitro

    SciTech Connect

    Li, G. Jane; Zhao Qiuqu; Zheng Wei . E-mail: wzheng@purdue.edu

    2005-06-01

    Manganese exposure alters iron homeostasis in blood and cerebrospinal fluid (CSF), possibly by acting on iron transport mechanisms localized at the blood-brain barrier and/or blood-CSF barrier. This study was designed to test the hypothesis that manganese exposure may change the binding affinity of iron regulatory proteins (IRPs) to mRNAs encoding transferrin receptor (TfR), thereby influencing iron transport at the blood-CSF barrier. A primary culture of choroidal epithelial cells was adapted to grow on a permeable membrane sandwiched between two culture chambers to mimic blood-CSF barrier. Trace {sup 59}Fe was used to determine the transepithelial transport of iron. Following manganese treatment (100 {mu}M for 24 h), the initial flux rate constant (K {sub i}) of iron was increased by 34%, whereas the storage of iron in cells was reduced by 58%, as compared to controls. A gel shift assay demonstrated that manganese exposure increased the binding of IRP1 and IRP2 to the stem loop-containing mRNAs. Consequently, the cellular concentrations of TfR proteins were increased by 84% in comparison to controls. Assays utilizing RT-PCR, quantitative real-time reverse transcriptase-PCR, and nuclear run off techniques showed that manganese treatment did not affect the level of heterogeneous nuclear RNA (hnRNA) encoding TfR, nor did it affect the level of nascent TfR mRNA. However, manganese exposure resulted in a significantly increased level of TfR mRNA and reduced levels of ferritin mRNA. Taken together, these results suggest that manganese exposure increases iron transport at the blood-CSF barrier; the effect is likely due to manganese action on translational events relevant to the production of TfR, but not due to its action on transcriptional, gene expression of TfR. The disrupted protein-TfR mRNA interaction in the choroidal epithelial cells may explain the toxicity of manganese at the blood-CSF barrier.

  5. Expression of prostanoid receptors in human ductus arteriosus.

    PubMed

    Leonhardt, Andreas; Glaser, Alexander; Wegmann, Markus; Schranz, Dietmar; Seyberth, Hannsjörg; Nüsing, Rolf

    2003-02-01

    1. Prostaglandins play a major role in maintaining ductal patency in utero. Ductal tone is regulated by both locally released and circulating vasodilatory prostaglandins. In infants with ductus arteriosus-dependent congenital heart disease, ductal patency is maintained by intravenous administration of prostaglandin (PG) E(1). Little information is available regarding the expression of prostaglandin receptors in man. 2. By means of RT-PCR and immunohistochemistry we studied the expression of the PGI(2) receptor (IP), the four different PGE(2) receptors (EP1, EP2, EP3 and EP4), and the receptors for thromboxane (Tx) A(2) (TP), PGD(2) (DP) and PGF(2alpha) (FP) in the ductus arteriosus of three newborn infants with ductus arteriosus-dependent congenital heart disease and intravenous infusion of PGE(1) and of one 8 month old child with a patent ductus arteriosus. 3. The EP3, EP4, FP, IP and TP receptor were markedly expressed at the mRNA and protein level, whereas the EP2 receptor was weakly expressed and the EP1 receptor was detected in two out of four tissue specimens only. The DP receptor was not detected in any of the samples. The most pronounced expression, which was located in the media of the ductus arteriosus, was observed for the EP4 and TP receptors followed by IP and FP receptor protein. 4. These data indicate that ductal patency during the infusion of PGE(1) in infants with ductus arteriosus-dependent congenital heart disease might be mediated by the EP4 and IP receptor. The data further suggest that a heterogeneous population of prostanoid receptors may contribute to the regulation of ductus arteriosus tone in humans. PMID:12598419

  6. Endotoxin suppresses rat hepatic low-density lipoprotein receptor expression.

    PubMed Central

    Liao, W; Rudling, M; Angelin, B

    1996-01-01

    Endotoxin induces hyperlipidaemia in experimental animals. In the current study, we investigated whether endotoxin alters hepatic low-density lipoprotein (LDL) receptor expression in rats. Endotoxin treatment suppressed hepatic LDL receptor expression in a dose- and time-dependent manner. Eighteen hours after intraperitoneal injection of increasing amounts of endotoxin, LDL receptor and its mRNA levels were determined by ligand blot and solution hybridization respectively. LDL receptor expression was inhibited by about 70% at a dose of 500 micrograms/100 g body weight. However, LDL receptor mRNA levels were markedly increased in all endotoxin-treated groups at this time point (by 83-136%; P < 0.001). Time-course experiments showed that LDL receptor expression was already reduced by 48% 4 h after endotoxin injection and was maximally reduced (by 63-65%) between 8 and 18 h. Changes in hepatic LDL receptor mRNA showed a different pattern. By 4 h after endotoxin injection, LDL receptor mRNA had decreased by 78% (P < 0.001). However, by 8 h after endotoxin injection, LDL receptor mRNA had returned to levels similar to controls, and 18 and 24 h after endotoxin injection, they were increased by about 60% (P < 0.05). Separation of plasma lipoproteins by FPLC demonstrated that endotoxin-induced changes in plasma triacylglycerols and cholesterol were due to accumulation of plasma apolipoprotein B-containing lipoproteins among very-low-density lipoprotein, intermediate-density lipoprotein and LDL. It is concluded that endotoxin suppresses hepatic LDL receptor expression in vivo in rats. PMID:8611169

  7. Elevated expression levels of androgen receptors and matrix metalloproteinase-2 and -9 in 30 cases of hepatocellular carcinoma compared with adjacent tissues as predictors of cancer invasion and staging

    PubMed Central

    ZHANG, YAN; SHEN, YUCHENG; CAO, BIN; YAN, AITING; JI, HAOMING

    2015-01-01

    The aim of the present study was to investigate the potential roles of the androgen receptor (AR) and matrix metalloproteinase (MMP)-2 and MMP-9 in hepatocellular carcinoma (HCC) tissues and whether their expression could be used as a predictor of the invasion and stage of cancer. The expression levels of AR, MMP-2 and MMP-9 in HCC tissues and tissues adjacent to the tumor were measured by immunohistochemical staining assay. The expression rates of AR, MMP-2 and MMP-9 in the HCC tissue were 76.67, 73.33 and 76.67%, respectively, all of which were significantly higher than those in the tissues adjacent to the tumor. The expression of these proteins represents the local invasion and stage. AR, MMP-2 and MMP-9 expression levels in HCC tissues have the potential to be employed as predictors of the progression of local cancer invasion and the tumor stage. PMID:25667651

  8. Developmental expression of retinoic acid receptors (RARs)

    PubMed Central

    Dollé, Pascal

    2009-01-01

    Here, I review the developmental expression features of genes encoding the retinoic acid receptors (RARs) and the 'retinoid X' or rexinoid receptors (RXRs). The first detailed expression studies were performed in the mouse over two decades ago, following the cloning of the murine Rar genes. These studies revealed complex expression features at all stages of post-implantation development, one receptor gene (Rara) showing widespread expression, the two others (Rarb and Rarg) with highly regionalized and/or cell type-specific expression in both neural and non-neural tissues. Rxr genes also have either widespread (Rxra, Rxrb), or highly-restricted (Rxrg) expression patterns. Studies performed in zebrafish and Xenopus demonstrated expression of Rar and Rxr genes (both maternal and zygotic), at early pre-gastrulation stages. The eventual characterization of specific enzymes involved in the synthesis of retinoic acid (retinol/retinaldehyde dehydrogenases), or the triggering of its catabolism (CYP26 cytochrome P450s), all of them showing differential expression patterns, led to a clearer understanding of the phenomenons regulated by retinoic acid signaling during development. Functional studies involving targeted gene disruptions in the mouse, and additional approaches such as dominant negative receptor expression in other models, have pinpointed the specific, versus partly redundant, roles of the RARs and RXRs in many developing organ systems. These pleiotropic roles are summarized hereafter in relationship to the receptors’ expression patterns. PMID:19471585

  9. Vitamin D Receptor Expression in Vitiligo

    PubMed Central

    Doss, Reham William; El-Rifaie, Abdel-Aziz; Gohary, Yasser M; Rashed, Laila A

    2015-01-01

    Background: Vitiligo is a progressive depigmenting disorder characterized by loss of functional melanocytes from the epidermis. The etiopathogenesis of vitiligo is still unclear. Vitamin D has stimulatory effects on melanocytes and acts through its nuclear Vitamin D receptor (VDR) on target cells. Aims and Objectives: The purpose of this study was to declare the role of Vitamin D in the pathogenesis of vitiligo. Materials and Methods: This case-control study included 30 vitiligo patients and 30 age, gender-matched healthy controls. Blood samples were withdrawn from the study subjects, and the serum 25(OH) D level was determined by an enzyme-linked immunosorbent assay technique. Serum 25(OH) D levels were divided into: Normal or sufficient (≥30 ng/ml), insufficient (< 30-> 20ng/ml), and deficient (≤20 ng/ml) levels. Skin biopsies were obtained from the depigmented lesions and clinically normal skin of vitiligo patients and from the controls, and VDR gene expression was determined using real-time polymerase chain reaction. Results: Only 10 patients with vitiligo (33.3%) had sufficient serum 25(OH) D levels (≥30 ng/ml), 12 patients (40%) had insufficient levels, and 8 patients (26.7%) had deficient levels. On the other hand, most of the controls (96.7%) had sufficient levels. The mean serum 25(OH) D level in patients was significantly decreased compared to controls (P < 0.001). The VDR-mRNA expression was also significantly decreased in lesional and nonlesional skin of patients compared to controls (P < 0.001, P < 0.001, respectively). Conclusion: Vitamin D deficiency influences the extent of vitiligo and could contribute to the pathogenesis of vitiligo through its immunomodulatory role and its role in melanogenesis. PMID:26677265

  10. Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: Expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies

    SciTech Connect

    Akamizu, Takashi; Ikuyama, Shoichiro; Saji, Motoyasu; Kosugi, Shinji; Kozak, C.; McBride, O.W.; Kohn, L.D. )

    1990-08-01

    A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, M{sub r} 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind {sup 125}I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3{prime} noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by throid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting autoantibodies, which actually increased TSH receptor mRNA levels.

  11. Cloning, chromosomal assignment, and regulation of the rat thyrotropin receptor: expression of the gene is regulated by thyrotropin, agents that increase cAMP levels, and thyroid autoantibodies.

    PubMed Central

    Akamizu, T; Ikuyama, S; Saji, M; Kosugi, S; Kozak, C; McBride, O W; Kohn, L D

    1990-01-01

    A rat thyrotropin (thyroid-stimulating hormone, TSH) receptor cDNA was isolated that encoded a protein of 764 amino acids, Mr 86,528. Transfection of the cDNA caused COS-7 cells to develop a TSH-sensitive adenylate cyclase response and the ability to bind 125I-labeled TSH; both activities were similar to those of rat FRTL-5 thyroid cells and not duplicated by lutropin. The gene represented by the cDNA was assigned to mouse chromosome 12 and human chromosome 14. Northern analyses identified two species of mRNA, 5.6 and 3.3 kilobases, in FRTL-5 thyroid cells; the transcripts appeared to differ only in the extent of their 3' noncoding sequences. There were minimal amounts of the two mRNAs in rat ovary, and neither was detected in RNA preparations from rat testis, liver, lung, brain, spleen, and FRT thyroid cells, which do not have a functional TSH receptor. TSH decreased both mRNA species 3- to 4-fold within 8 hr in FRTL-5 thyroid cells; down-regulation was dependent on TSH concentration and duplicated by forskolin, cholera toxin, or 8-bromo-cAMP but not by a phorbol ester. Down-regulation was also duplicated by thyroid-stimulating autoantibodies, which increased cAMP levels, but not by thyrotropin binding-inhibiting auto-antibodies, which actually increased TSH receptor mRNA levels. Images PMID:1696008

  12. Cloning, constitutive activity and expression profiling of two receptors related to relaxin receptors in Drosophila melanogaster.

    PubMed

    Van Hiel, Matthias B; Vandersmissen, Hans Peter; Proost, Paul; Vanden Broeck, Jozef

    2015-06-01

    Leucine-rich repeat containing G protein-coupled receptors (LGRs) comprise a cluster of transmembrane proteins, characterized by the presence of a large N-terminal extracellular domain. This receptor group can be classified into three subtypes. Belonging to the subtype C LGRs are the mammalian relaxin receptors LGR7 (RXFP1) and LGR8 (RXFP2), which mediate important reproductive and other processes. We identified two related receptors in the genome of the fruit fly and cloned their open reading frames into an expression vector. Interestingly, dLGR3 demonstrated constitutive activity at very low doses of transfected plasmid, whereas dLGR4 did not show any basal activity. Both receptors exhibited a similar expression pattern during development, with relatively high transcript levels during the first larval stage. In addition, both receptors displayed higher expression in male adult flies as compared to female flies. Analysis of the tissue distribution of both receptor transcripts revealed a high expression of dLGR3 in the female fat body, while the expression of dLGR4 peaked in the midgut of both the wandering and adult stage. PMID:25064813

  13. Reduced VLDL clearance in Apoe(-/-)Npc1(-/-) mice is associated with increased Pcsk9 and Idol expression and decreased hepatic LDL-receptor levels.

    PubMed

    Ishibashi, Minako; Masson, David; Westerterp, Marit; Wang, Nan; Sayers, Scott; Li, Rong; Welch, Carrie L; Tall, Alan R

    2010-09-01

    Niemann-Pick type C1 (NPC1) promotes the transport of LDL receptor (LDL-R)-derived cholesterol from late endosomes/lysosomes to other cellular compartments. NPC1-deficient cells showed impaired regulation of liver_X receptor (LXR) and sterol regulatory element-binding protein (SREBP) target genes. We observed that Apoe(-/-)Npc1(-/-) mice displayed a marked increase in total plasma cholesterol mainly due to increased VLDL, reflecting decreased clearance. Although nuclear SREBP-2 and Ldlr mRNA levels were increased in Apoe(-/-)Npc1(-/-) liver, LDL-R protein levels were decreased in association with marked induction of proprotein convertase subtilisin/kexin type 9 (Pcsk9) and inducible degrader of the LDL-R (Idol), both known to promote proteolytic degradation of LDL-R. While Pcsk9 is known to be an SREBP-2 target, marked upregulation of IDOL in Apoe(-/-)Npc1(-/-) liver was unexpected. However, several other LXR target genes also increased in Apoe(-/-)Npc1(-/-) liver, suggesting increased synthesis of endogenous LXR ligands secondary to activation of sterol biosynthesis. In conclusion, we demonstrate that NPC1 deficiency has a major impact on VLDL metabolism in Apoe(-/-) mice through modulation of hepatic LDL-R protein levels. In contrast to modest induction of hepatic IDOL with synthetic LXR ligands, a striking upregulation of IDOL in Apoe(-/-)Npc1(-/-) mice could indicate a role of endogenous LXR ligands in regulation of hepatic IDOL. PMID:20562239

  14. Repertoire of Chemokine Receptor Expression in the Female Genital Tract

    PubMed Central

    Patterson, Bruce K.; Landay, Alan; Andersson, Jan; Brown, Clark; Behbahani, Homira; Jiyamapa, Dan; Burki, Zareefa; Stanislawski, Donna; Czerniewski, Mary Ann; Garcia, Patricia

    1998-01-01

    Sexually transmitted diseases, genital ulcer disease, and progesterone therapy increase susceptibility to lentivirus transmission. Infection of cells by human immunodeficiency virus (HIV) is dependent on expression of specific chemokine receptors known to function as HIV co-receptors. Quantitative kinetic reverse transcription-polymerase chain reaction was developed to determine the in vivo expression levels of CCR5, CXCR4, CCR3, CCR2b, and the cytomegalovirus-encoded US28 in peripheral blood mononuclear cells and cervical biopsies from 12 women with and without sexually transmitted diseases, genital ulcer disease, and progesterone-predominant conditions. Our data indicate that CCR5 is the major HIV co-receptor expressed in the female genital tract, and CXCR4 is the predominantly expressed HIV co-receptor in peripheral blood. CCR5 mRNA expression in the ectocervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In peripheral blood, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in cervical tissue despite expression in peripheral blood mononuclear cells from five individuals. CCR5 was significantly increased (p < 0.02) in biopsies from women with sexually transmitted diseases and others who were progesterone predominant. In vitro studies demonstrate that progesterone increases CCR5, CXCR4, and CCR3 expression and decreases CCR2b expression in lymphocytes and monocytes/macrophages. Characterization of chemokine receptors at the tissue level provides important information in identifying host determinants of HIV-1 transmission. PMID:9708808

  15. Role of polymorphic Fc gamma receptor IIIa and EGFR expression level in cetuximab mediated, NK cell dependent in vitro cytotoxicity of head and neck squamous cell carcinoma cells.

    PubMed

    López-Albaitero, Andrés; Lee, Steve C; Morgan, Sarah; Grandis, Jennifer R; Gooding, William E; Ferrone, Soldano; Ferris, Robert L

    2009-11-01

    Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10-20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients' differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcgammaR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcgammaR polymorphism. Effector cells expressing the FcgammaR IIIa-158 VV allele were significantly (P < 0.0001) more effective than those expressing FcgammaR IIIa VF and FF [corrected] alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P < 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcgammaR IIIa 158 FF expressing NK cells. The importance of the FcgammaR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient

  16. Role of polymorphic Fc gamma receptor IIIa and EGFR expression level in cetuximab mediated, NK cell dependent in vitro cytotoxicity of head and neck squamous cell carcinoma cells

    PubMed Central

    López-Albaitero, Andrés; Lee, Steve C.; Morgan, Sarah; Grandis, Jennifer R.; Gooding, William E.; Ferrone, Soldano

    2012-01-01

    Immunotherapy with the EGFR-specific mAb cetuximab is clinically effective in 10–20% of patients with squamous cell carcinoma of the head and neck (SCCHN). Little information is available about the mechanism(s) underlying patients’ differential clinical response to cetuximab-based immunotherapy, although this information may contribute to optimizing the design of cetuximab-based immunotherapy. Our understanding of these mechanisms would benefit from the characterization of the variables which influence the extent of cell dependent-lysis of SCCHN cells incubated with cetuximab in vitro. Therefore, in this study we have investigated the role of FcγR IIIa-158 genotype expressed by effector NK cells, cetuximab concentration, and EGFR expression level by SCCHN cells in the extent of their in vitro lysis and in the degree of NK cell activation. PBMC or purified CD56+ NK cells genotyped at IIIa codon 158 and SCCHN cell lines expressing different levels of EGFR have been used as effectors and targets, respectively, in antibody dependent cellular cytotoxicity (ADCC) assays. Furthermore, supernatants from ADCC assays were analyzed for cytokine and chemokine levels using multiplexed ELISA. We found that the extent of lysis of SCCHN cells was influenced by the EGFR expression level, cetuximab concentration, and FcγR polymorphism. Effector cells expressing the FcγR IIIa-158 VV allele were significantly (P < 0.0001) more effective than those expressing FcγR IIIa VF and VV alleles in mediating lysis of SCCHN cells expressed higher levels of the activation markers CD69 and CD107a, and secreted significantly (P < 0.05) larger amounts of inflammatory cytokines and chemokines. IL-2 or IL-15 treatment increased cetuximab-mediated ADCC by poor binding FcγR IIIa 158 FF expressing NK cells. The importance of the FcγR IIIa-158 polymorphism in cytotoxicity of SCCHN cells by NK cells supports a potential role for immune activation and may explain patient variability of cetuximab

  17. Functional expression of purinergic P2 receptors and transient receptor potential channels by the human urothelium

    PubMed Central

    Shabir, Saqib; Cross, William; Kirkwood, Lisa A.; Pearson, Joanna F.; Appleby, Peter A.; Walker, Dawn; Eardley, Ian

    2013-01-01

    In addition to its role as a physical barrier, the urothelium is considered to play an active role in mechanosensation. A key mechanism is the release of transient mediators that activate purinergic P2 receptors and transient receptor potential (TRP) channels to effect changes in intracellular Ca2+. Despite the implied importance of these receptors and channels in urothelial tissue homeostasis and dysfunctional bladder disease, little is known about their functional expression by the human urothelium. To evaluate the expression and function of P2X and P2Y receptors and TRP channels, the human ureter and bladder were used to separate urothelial and stromal tissues for RNA isolation and cell culture. RT-PCR using stringently designed primer sets was used to establish which P2 and TRP species were expressed at the transcript level, and selective agonists/antagonists were used to confirm functional expression by monitoring changes in intracellular Ca2+ and in a scratch repair assay. The results confirmed the functional expression of P2Y4 receptors and excluded nonexpressed receptors/channels (P2X1, P2X3, P2X6, P2Y6, P2Y11, TRPV5, and TRPM8), while a dearth of specific agonists confounded the functional validation of expressed P2X2, P2X4, P2Y1, P2Y2, TRPV2, TRPV3, TRPV6 and TRPM7 receptors/channels. Although a conventional response was elicited in control stromal-derived cells, the urothelial cell response to well-characterized TRPV1 and TRPV4 agonists/antagonists revealed unexpected anomalies. In addition, agonists that invoked an increase in intracellular Ca2+ promoted urothelial scratch repair, presumably through the release of ATP. The study raises important questions about the ligand selectivity of receptor/channel targets expressed by the urothelium. These pathways are important in urothelial tissue homeostasis, and this opens the possibility of selective drug targeting. PMID:23720349

  18. Stable Expression and Characterization of an Optimized Mannose Receptor

    PubMed Central

    Vigerust, David J; Vick, Sherell; Shepherd, Virginia L

    2015-01-01

    The mannose receptor (MR) is a macrophage surface receptor that recognizes pathogen associated molecular patterns (PAMPs) from a diverse array of bacterial, fungal and viral pathogens. Functional studies of the MR are hampered by the scarcity of human cell lines that express the receptor. Current model systems available for the study of MR biology often demonstrate low levels of expression and do not retain many of the classical MR properties. Although several laboratories have reported transient and stable expression of MR from plasmids, preliminary data from our laboratory suggests that these plasmids produce a protein that lacks critical domains and is often not stable over time. In this current report we describe the generation and characterization of a novel human codon-optimized system for transient and stable MR expression. Rare codons and sequences that contribute to mRNA instability were modified to produce mRNA that is qualitatively and quantitatively improved. Confocal imaging of the transient and stably expressed optimized receptor demonstrates a distribution consistent with previous reports. To demonstrate the functional characteristics of the optimized receptor, we further show that the introduction of codon-optimized MR plasmid can confer MR-associated phagocytosis of S. aureus to non-phagocytic HeLa cells. We show that three molecules participate in the engagement and internalization of S. aureus. MR was found to colocalize with Toll-like receptor 2 (TLR2) and Rab5 following exposure to pHrodo-stained S. aureus, suggesting cooperation among the three molecules to engage and internalize the bacterial particle. This study describes a transfection capable, optimized MR receptor with functional characteristics similar to the wild type receptor and further demonstrates a new system for the continued study of MR biology and function. PMID:26581716

  19. Sinonasal Leiomyoma With Estrogen Receptor Expression.

    PubMed

    Kim, Jong Seung; Shin, Jin Yong; Kwon, Sam Hyun

    2015-09-01

    Leiomyoma is an extremely rare tumor in sinonasal area. The reason for this is due to minimal amount of the smooth muscle in the area. The origin of this tumor is not clear and its etiology has not been proven in the literature. A 58-year-old woman who experienced nasal obstruction and epiphora visited our clinic. A huge mass was noted in right nasal cavity originating from the lacrimal bone area. The authors conducted endoscopic sinus surgery and obtained the specimen. Immunochemistry showed leiomyoma in the nasal cavity, which expressed estrogen receptor. There was no progesterone receptor expressed. The authors describe a sinonasal leiomyoma with estrogen receptors, not ever reported in previous article. PMID:26355987

  20. Expression of Angiotensin II Receptor-1 in Human Articular Chondrocytes

    PubMed Central

    Kawakami, Yuki; Matsuo, Kosuke; Murata, Minako; Yudoh, Kazuo; Nakamura, Hiroshi; Shimizu, Hiroyuki; Beppu, Moroe; Inaba, Yutaka; Saito, Tomoyuki; Kato, Tomohiro; Masuko, Kayo

    2012-01-01

    Background. Besides its involvement in the cardiovascular system, the renin-angiotensin-aldosterone (RAS) system has also been suggested to play an important role in inflammation. To explore the role of this system in cartilage damage in arthritis, we investigated the expression of angiotensin II receptors in chondrocytes. Methods. Articular cartilage was obtained from patients with osteoarthritis, rheumatoid arthritis, and traumatic fractures who were undergoing arthroplasty. Chondrocytes were isolated and cultured in vitro with or without interleukin (IL-1). The expression of angiotensin II receptor types 1 (AT1R) and 2 (AT2R) mRNA by the chondrocytes was analyzed using reverse transcription-polymerase chain reaction (RT-PCR). AT1R expression in cartilage tissue was analyzed using immunohistochemistry. The effect of IL-1 on AT1R/AT2R expression in the chondrocytes was analyzed by quantitative PCR and flow cytometry. Results. Chondrocytes from all patient types expressed AT1R/AT2R mRNA, though considerable variation was found between samples. Immunohistochemical analysis confirmed AT1R expression at the protein level. Stimulation with IL-1 enhanced the expression of AT1R/AT2R mRNA in OA and RA chondrocytes. Conclusions. Human articular chondrocytes, at least partially, express angiotensin II receptors, and IL-1 stimulation induced AT1R/AT2R mRNA expression significantly. PMID:23346400

  1. Transgenic silkworms expressing human insulin receptors for evaluation of therapeutically active insulin receptor agonists.

    PubMed

    Matsumoto, Yasuhiko; Ishii, Masaki; Ishii, Kenichi; Miyaguchi, Wataru; Horie, Ryo; Inagaki, Yoshinori; Hamamoto, Hiroshi; Tatematsu, Ken-ichiro; Uchino, Keiro; Tamura, Toshiki; Sezutsu, Hideki; Sekimizu, Kazuhisa

    2014-12-12

    We established a transgenic silkworm strain expressing the human insulin receptor (hIR) using the GAL4/UAS system. Administration of human insulin to transgenic silkworms expressing hIR decreased hemolymph sugar levels and facilitated Akt phosphorylation in the fat body. The decrease in hemolymph sugar levels induced by injection of human insulin in the transgenic silkworms expressing hIR was blocked by co-injection of wortmannin, a phosphoinositide 3-kinase inhibitor. Administration of bovine insulin, an hIR ligand, also effectively decreased sugar levels in the transgenic silkworms. These findings indicate that functional hIRs that respond to human insulin were successfully induced in the transgenic silkworms. We propose that the humanized silkworm expressing hIR is useful for in vivo evaluation of the therapeutic activities of insulin receptor agonists. PMID:25449269

  2. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-07-15

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  3. Purification of a rat neurotensin receptor expressed in Escherichia coli.

    PubMed Central

    Tucker, J; Grisshammer, R

    1996-01-01

    A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress. PMID:8760379

  4. P2×7 purinergic signaling in dilated cardiomyopathy induced by auto-immunity against muscarinic M2 receptors: autoantibody levels, heart functionality and cytokine expression

    PubMed Central

    Martinez, Camila Guerra; Zamith-Miranda, Daniel; da Silva, Marcia Gracindo; Ribeiro, Karla Consort; Brandão, Izaíra Trincani; Silva, Celio Lopes; Diaz, Bruno Lourenço; Bellio, Maria; Persechini, Pedro Muanis; Kurtenbach, Eleonora

    2015-01-01

    Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems, and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM. PMID:26592184

  5. Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles.

    PubMed

    Satchell, Leanne; Glister, Claire; Bleach, Emma C; Glencross, Richard G; Bicknell, Andrew B; Dai, Yanzhenzi; Anand-Ivell, Ravinder; Ivell, Richard; Knight, Philip G

    2013-05-01

    Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary, but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna cell (TIC) and granulosa cell compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than granulosa cell and increased progressively during follicle maturation with INSL3 peaking in large (11-18 mm) estrogen-active follicles and RXFP2 peaking in 9- to 10-mm follicles before declining in larger (11-18 mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly autocrine/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant preovulatory follicle, is detectable in peripheral blood of cattle, and expression is down-regulated during luteinization induced by the preovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, while raising doubts about its potential contribution to CL function. PMID:23546605

  6. Roosters affected by epididymal lithiasis present local alteration in vitamin D3, testosterone and estradiol levels as well as estrogen receptor 2 (beta) expression.

    PubMed

    Oliveira, André G; Dornas, Rubem A P; Praes, Lílian C; Hess, Rex A; Mahecha, Germán A B; Oliveira, Cleida A

    2011-09-01

    Epididymal lithiasis is a reproductive dysfunction of roosters that is associated with loss of fertility and is characterized by the formation of calcium stones in the lumen of the efferent ductules of the epididymal region. The efferent ductules of birds are responsible for the reabsorption of the fluid coming from the testis as well as luminal calcium. It has been hypothesized that the epididymal stone formation may be related to the impairment of local fluid or calcium homeostasis, which depends on hormones such as estradiol (E(2)). Therefore, this study aimed to investigate possible alterations in the expression of ERα (ESR1) and ERβ (ESR2) in the epididymal region of roosters affected by epididymal lithiasis. The study was performed by immunohistochemistry and western blotting assays. In addition, the concentrations of E(2), vitamin D3, and testosterone, which are also key hormones in maintenance of calcium homeostasis, were determined in the plasma and epididymal region, by ELISA. It was observed that ESR2 expression is increased in all segments of the epididymal region of affected roosters, whereas ESR1 levels are not altered. Moreover, the hormone concentration profiles were changed, as in the epididymal region of roosters with lithiasis the E(2) levels were increased and vitamin D3 as well as testosterone concentrations were significantly decreased. These results suggest that a hormonal imbalance may be involved with the origin and progression of the epididymal lithiasis, possibly by affecting the local fluid or calcium homeostasis. PMID:21670126

  7. Effect of neonatal or adult heat acclimation on plasma fT3 level, testicular thyroid receptors expression in male rats and testicular steroidogenesis in vitro in response to triiodothyronine treatment.

    PubMed

    Kurowicka, B; Chrusciel, M; Zmijewska, A; Kotwica, G

    2016-01-01

    This study aimed to evaluate the effect of heat acclimation of neonatal and adult rats on their testes response to in vitro treatment with triiodothyronine (T3). Four groups of rats were housed from birth as: 1) control (CR) at 20°C for 90 days, 2) neonatal heat-acclimated (NHA) at 34°C for 90 days, 3) adult heat-acclimated (AHA) at 20°C for 45 days followed by 45 days at 34°C and 4) de-acclimated (DA) at 34°C for 45 days followed by 45 days at 20°C. Blood plasma and both testes were harvested from 90-day old rats. Testicular slices were then submitted to in vitro treatment with T3 (100 ng/ml) for 8 h. Plasma fT3 level was lower in AHA, NHA and DA groups than in CR group. Basal thyroid hormone receptor α1 (Thra1) expression was higher in testes of NHA and DA and β1 receptor (Thrb1) in DA rats vs. other groups. In the in vitro experiment, T3: 1) decreased Thra1 expression in all groups and Thrb1 in DA group, 2) increased Star expression in CR, NHA and DA groups, and Hsd17b3 expression in NHA group, 3) decreased the expression of Cyp11a1 in NHA and DA groups, and Cyp19a1 in all the groups, 4) did not affect the activity of steroidogenic enzymes and steroid secretion (A4, T, E2) in all the groups. These results indicate, that heat acclimation of rats, depending on their age, mainly affects the testicular expression of steroidogenic enzymes in response to short-lasting treatment with T3. PMID:27487513

  8. Differential Expression of Motilin Receptor in Various Parts of Gastrointestinal Tract in Dogs

    PubMed Central

    He, Yu; Wang, Hui; Yang, DongYan; Wang, ChengYan; Yang, LanLan; Jin, Chunxiang

    2015-01-01

    Objectives. The presence of motilin receptor in the GI tract of different animal species has been verified. However, the quantitation of motilin receptor expression in different regions of the GI tract remains unclear. The aim of this study was to investigate the expression of motilin receptor in the GI tract and semiquantitatively compare the expression difference in different GI regions in dogs. Methods. Antrum, duodenum, jejunum, ileum, proximal colon, middle colon, and distal colon were obtained from various parts of the GI tract of six sacrificed dogs. The distribution of motilin receptor was determined by immunohistochemistry. The expression levels of motilin receptor mRNA in different regions were measured by RT-PCR. Results. Motilin receptor was expressed throughout the GI tract in dogs. Multiple comparisons of the mean motilin receptor mRNA expression among various regions were significant (P < 0.05). Motilin receptor mRNA was extensively expressed in duodenum, followed by ileum, jejunum, proximal colon, antrum, middle colon, and distal colon. Immunohistochemistry revealed that motilin receptor immunoreactivity was observed only in the enteric nervous system. Conclusion. Motilin receptor is expressed differentially along the GI tract in dogs. The significantly high expression of motilin receptor mRNA is found in the duodenum. PMID:25918525

  9. Expression of Estrogen Receptor α in the Mouse Cerebral Cortex

    PubMed Central

    Dietrich, Alicia K.; Humphreys, Gwendolyn I.; Nardulli, Ann M.

    2015-01-01

    Although estrogen receptor alpha (ERα) and 17β-estradiol play critical roles in protecting the cerebral cortex from ischemia-induced damage, there has been some controversy about the expression of ERα in this region of the brain. We have examined ERα mRNA and protein levels in the cerebral cortices of female mice at postnatal days 5 and 17 and at 4, 13, and 18 months of age. We found that although ERα transcript levels declined from postnatal day 5 through 18 months of age, ERα protein levels remained stable. Importantly, expression of the E2-regulated progesterone receptor gene was sustained in younger and in older females suggesting that age-related changes in estrogen responsiveness in the cerebral cortex are not due to the absence of ERα protein. PMID:25700604

  10. Detection of CXCR2 cytokine receptor surface expression using immunofluorescence.

    PubMed

    Lam, Clarissa; Pavel, Mahmud Arif; Kashyap, Parul; Salehi-Najafabadi, Zahra; Valentino, Victoria; Yu, Yong

    2014-01-01

    The interleukin-8 (IL-8, CXCL8) chemokine, also known as the neutrophil chemotactic factor, is a cytokine that plays a key role in inflammatory response, cell proliferation, migration, and survival. IL-8 expression is increased not only in inflammatory disorders, but also in many types of cancer, including prostate cancer. IL-8 acts as a ligand for the C-X-C chemokine receptor 2 (CXCR2) protein present on the cell plasma membrane. Binding of the IL-8 ligand to the CXCR2 receptor results in an intracellular signaling pathway mediated by GTP binding proteins coupled to the receptor itself. Knowledge of the CXCR2 expression levels facilitates the understanding of the role and function of IL-8. In this chapter, we describe a protocol that uses the immunofluorescence method and confocal microscopy to analyze the CXCR2 surface expression in human prostate cancer cells. However, this protocol is easily adaptable to analyze the surface expression of other cytokine receptors in different cell types. PMID:24908306

  11. Expression of Hepcidin and Ferroportin in the Placenta, and Ferritin and Transferrin Receptor 1 Levels in Maternal and Umbilical Cord Blood in Pregnant Women with and without Gestational Diabetes

    PubMed Central

    Yang, Anqiang; Zhao, Jun; Lu, Minhua; Gu, Ying; Zhu, Yunlong; Chen, Daozhen; Fu, Jinyan

    2016-01-01

    Background: Regulation of iron transfer from mother to fetus via the placenta is not fully understood and the relationship between stored iron status in the mothers’ serum and gestational diabetes (GDM) in case–control studies is controversial. The present study aimed to detect circulating soluble transferrin receptor (sTfR) and ferritin levels in maternal and umbilical cord blood. We also examined the expression of hepcidin (Hep), transferrin receptor (TfR1), and ferroportin (FPN) in the placenta in pregnant women with and without GDM at full term. Methods: Eighty-two women participated (42 with GDM and 40 without GDM [controls]). Maternal samples were collected at 37–39 weeks’ gestation. Umbilical cord blood was collected at birth. Ferritin and sTfR levels in maternal serum and umbilical cord blood, and Hep, TfR1, and FPN protein expression in plac enta were compared between the GDM and non-GDM groups. Serum ferritin (SF) was measured by electrochemiluminescence assay and sTfR was measured by ELISA. Hep, TfR1, and FPN expression was measured by immunohistochemistry. Results: Maternal serum sTfR levels were significantly elevated in the GDM group compared with the non-GDM group (p = 0.003). SF levels in cord blood in the GDM group were significantly higher than those in the non-GDM group (p = 0.003). However, maternal hemoglobin and SF, and umbilical cord sTfR levels were not different between the groups. In placental tissue, FPN expression was higher and hepcidin expression was lower in the GDM group compared with the non-GDM group (p = 0.000 and p = 0.044, respectively). There was no significant difference in TfR1 between the groups (p = 0.898). Conclusions: Women with GDM transport iron more actively than those without GDM at term pregnancy. Maternal iron metabolism in GDM may play a role in fetal/placental iron demand and in the overall outcome of pregnancy. PMID:27483296

  12. Kinin receptor expression during Staphylococcus aureus infection

    PubMed Central

    Bengtson, Sara H.; Phagoo, Stephen B.; Norrby-Teglund, Anna; Påhlman, Lisa; Mörgelin, Matthias; Zuraw, Bruce L.; Leeb-Lundberg, L. M. Fredrik; Herwald, Heiko

    2006-01-01

    An inappropriate host response to invading bacteria is a critical parameter that often aggravates the outcome of an infection. Staphylococcus aureus is a major human Gram-positive pathogen that causes a wide array of community- and hospital-acquired diseases ranging from superficial skin infections to severe conditions such as staphylococcal toxic shock. Here we find that S aureus induces inflammatory reactions by modulating the expression and response of the B1 and B2 receptors, respectively. This process is initiated by a chain of events, involving staphylococcal-induced cytokine release from monocytes, bacteria-triggered contact activation, and conversion of bradykinin to its metabolite desArg9bradykinin. The data of the present study implicate an important and previously unknown role for kinin receptor regulation in S aureus infections. PMID:16735595

  13. TSH-receptor-expressing fibrocytes and thyroid-associated ophthalmopathy.

    PubMed

    Smith, Terry J

    2015-03-01

    Thyroid-associated ophthalmopathy (TAO) is a vexing and undertreated ocular component of Graves disease in which orbital tissues undergo extensive remodelling. My colleagues and I have introduced the concept that fibrocytes expressing the haematopoietic cell antigen CD34 (CD34(+) fibrocytes), which are precursor cells of bone-marrow-derived monocyte lineage, express the TSH receptor (TSHR). These cells also produce several other proteins whose expression was traditionally thought to be restricted to the thyroid gland. TSHR-expressing fibrocytes in which the receptor is activated by its ligand generate extremely high levels of several inflammatory cytokines. Acting in concert with TSHR, the insulin-like growth factor 1 receptor (IGF-1R) expressed by orbital fibroblasts and fibrocytes seems to be necessary for TSHR-dependent cytokine production, as anti-IGF-1R blocking antibodies attenuate these proinflammatory actions of TSH. Furthermore, circulating fibrocytes are highly abundant in patients with TAO and seem to infiltrate orbital connective tissues, where they might transition to CD34(+) fibroblasts. My research group has postulated that the infiltration of fibrocytes into the orbit, their unique biosynthetic repertoire and their proinflammatory and profibrotic phenotype account for the characteristic properties exhibited by orbital connective tissues that underlie susceptibility to TAO. These insights, which have emerged in the past few years, might be of use in therapeutically targeting pathogenic orbit-infiltrating fibrocytes selectively by utilizing novel biologic agents that interfere with TSHR and IGF-1R signalling. PMID:25560705

  14. Dopamine receptor expression and function in corticotroph pituitary tumors.

    PubMed

    Pivonello, Rosario; Ferone, Diego; de Herder, Wouter W; Kros, Johan M; De Caro, Maria Laura Del Basso; Arvigo, Marica; Annunziato, Lucio; Lombardi, Gaetano; Colao, Annamaria; Hofland, Leo J; Lamberts, Steven W J

    2004-05-01

    The role of dopamine agonist treatment in corticotroph pituitary tumors is controversial. The aim of this study was to evaluate D(2) receptor expression in 20 corticotroph pituitary tumors and to correlate it to the in vitro effect of dopamine agonists on ACTH secretion and the in vivo effect of short-term cabergoline treatment on cortisol secretion. D(2) expression was evaluated by receptor-ligand binding, immunohistochemistry, and RT-PCR. A 50% or more decrease in daily urinary cortisol levels was considered a significant clinical response. At receptor-ligand binding, specific binding of [(125)I]epidepride was found in 80% of cases. At immunohistochemistry, specific D(2) immunostaining was found in 75% of cases. D(2) expression was found in 83.3% of cases (D(2long) in 40%, D(2short) in 20%, and both in 40%) by RT-PCR. Significant in vitro inhibition of ACTH secretion was found in 100% of D(2)-positive cases, but not in 100% of D(2)-negative cases by either bromocriptine or cabergoline. A significant in vivo inhibition of cortisol secretion after 3-month cabergoline treatment was found in 60%, although a normalization of cortisol secretion was found in 40% of cases. All cabergoline-responsive cases were associated with D(2) expression, whereas all noncabergoline-responsive cases but one were not associated with D(2) expression. In conclusion, functional D(2) receptors were expressed in approximately 80% of corticotroph pituitary tumors. The effectiveness of cabergoline in normalizing cortisol secretion in 40% of cases supports its therapeutic use in the management of Cushing's disease. PMID:15126577

  15. Expression of glucocorticoid and progesterone nuclear receptor genes in archival breast cancer tissue

    PubMed Central

    Smith, Robert A; Lea, Rod A; Curran, Joanne E; Weinstein, Stephen R; Griffiths, Lyn R

    2003-01-01

    Background Previous studies in our laboratory have shown associations of specific nuclear receptor gene variants with sporadic breast cancer. In order to investigate these findings further, we conducted the present study to determine whether expression levels of the progesterone and glucocorticoid nuclear receptor genes vary in different breast cancer grades. Methods RNA was extracted from paraffin-embedded archival breast tumour tissue and converted into cDNA. Sample cDNA underwent PCR using labelled primers to enable quantitation of mRNA expression. Expression data were normalized against the 18S ribosomal gene multiplex and analyzed using analysis of variance. Results Analysis of variance indicated a variable level of expression of both genes with regard to breast cancer grade (P = 0.00033 for glucocorticoid receptor and P = 0.023 for progesterone receptor). Conclusion Statistical analysis indicated that expression of the progesterone nuclear receptor is elevated in late grade breast cancer tissue. PMID:12559052

  16. Effects of chromium-enriched Bacillus subtilis KT260179 supplementation on growth performance, caecal microbiology, tissue chromium level, insulin receptor expression and plasma biochemical profile of mice under heat stress.

    PubMed

    Yang, Jiajun; Xu, Yayuan; Qian, Kun; Zhang, Wei; Wu, Dong; Wang, Chonglong

    2016-03-14

    The aim of this study was to investigate the effect of providing supplementary Cr-enriched Bacillus subtilis (CEBS) to mice with regard to their growth performance, caecal microbiology, tissue Cr concentration, insulin receptor (IR) expression and plasma biochemical profile. A total of ninety-six Kunming strain mice were allocated to four different groups: control, CEBS, inorganic Cr and B. subtilis. After 15 d of treatment, mice that received CEBS or normal B. subtilis had higher body weights than control mice, and after 30 d mice given either CEBS or B. subtilis had greater body weights than control mice or those given inorganic Cr. The concentration of Cr in tissues (heart, liver, spleen, kidney and skeletal muscle) increased after CEBS supplementation. B. subtilis and CEBS supplementation caused a significant increase in the numbers of Lactobacillus and Bifidobacterium in the caecum, whereas the numbers of Escherichia coli and Staphylococcus decreased significantly compared with the control. The levels of IR RNA and protein in skeletal muscles increased significantly. Plasma glucose, total cholesterol, TAG and LDL-cholesterol levels declined significantly in the CEBS group compared with the control group, whereas plasma insulin and HDL-cholesterol levels increased significantly. In conclusion, CEBS supplementation enhanced the regulation of body growth, increased tissue organic Cr concentrations, altered caecal microbiota and enhanced IR expression to produce significant changes in plasma biochemistry. PMID:26758987

  17. Striatal adenosine-cannabinoid receptor interactions in rats over-expressing adenosine A2A receptors.

    PubMed

    Chiodi, Valentina; Ferrante, Antonella; Ferraro, Luca; Potenza, Rosa Luisa; Armida, Monica; Beggiato, Sarah; Pèzzola, Antonella; Bader, Michael; Fuxe, Kjell; Popoli, Patrizia; Domenici, Maria Rosaria

    2016-03-01

    Adenosine A2A receptors (A2 A Rs) and cannabinoid CB1 receptors (CB1 Rs) are highly expressed in the striatum, where they functionally interact and form A2A /CB1 heteroreceptor complexes. We investigated the effects of CB1 R stimulation in a transgenic rat strain over-expressing A2 A Rs under the control of the neural-specific enolase promoter (NSEA2A rats) and in age-matched wild-type (WT) animals. The effects of the CB1 R agonist WIN 55,212-2 (WIN) were significantly lower in NSEA2A rats than in WT animals, as demonstrated by i) electrophysiological recordings of synaptic transmission in corticostriatal slices; ii) the measurement of glutamate outflow from striatal synaptosomes and iii) in vivo experiments on locomotor activity. Moreover, while the effects of WIN were modulated by both A2 A R agonist (CGS 21680) and antagonists (ZM 241385, KW-6002 and SCH-442416) in WT animals, the A2 A R antagonists failed to influence WIN-mediated effects in NSEA2A rats. The present results demonstrate that in rats with genetic neuronal over-expression of A2 A Rs, the effects mediated by CB1 R activation in the striatum are significantly reduced, suggesting a change in the stoichiometry of A2A and CB1 receptors and providing a strategy to dissect the involvement of A2 A R forming or not forming heteromers in the modulation of striatal functions. These findings add additional evidence for the existence of an interaction between striatal A2 A Rs and CB1 Rs, playing a fundamental role in the regulation of striatal functions. We studied A2A -CB1 receptor interaction in transgenic rats over-expressing adenosine A2A receptors under the control of the neuron-specific enolase promoter (NSEA2A ). In these rats, we demonstrated a reduced effect of the CB1 receptor agonist WIN 55,212-2 in the modulation of corticostriatal synaptic transmission and locomotor activity, while CB1 receptor expression level did not change with respect to WT rats. A reduction in the expression of A2A -CB1

  18. Cell-Free Expression of G Protein-Coupled Receptors.

    PubMed

    Segers, Kenneth; Masure, Stefan

    2015-01-01

    The large-scale production of recombinant G protein-coupled receptors (GPCRs) is one of the major bottlenecks that hamper functional and structural studies of this important class of integral membrane proteins. Heterologous overexpression of GPCRs often results in low yields of active protein, usually due to a combination of several factors, such as low expression levels, protein insolubility, host cell toxicity, and the need to use harsh and often denaturing detergents (e.g., SDS, LDAO, OG, and DDM, among others) to extract the recombinant receptor from the host cell membrane. Many of these problematic issues are inherently linked to cell-based expression systems and can therefore be circumvented by the use of cell-free systems. In this unit, we provide a range of protocols for the production of GPCRs in a cell-free expression system. Using this system, we typically obtain GPCR expression levels of ∼1 mg per ml of reaction mixture in the continuous-exchange configuration. Although the protocols in this unit have been optimized for the cell-free expression of GPCRs, they should provide a good starting point for the production of other classes of membrane proteins, such as ion channels, aquaporins, carrier proteins, membrane-bound enzymes, and even large molecular complexes. PMID:26237676

  19. Triggering Receptor Expressed on Myeloid Cells in Cutaneous Melanoma.

    PubMed

    Nguyen, Austin Huy; Koenck, Carleigh; Quirk, Shannon K; Lim, Victoria M; Mitkov, Mario V; Trowbridge, Ryan M; Hunter, William J; Agrawal, Devendra K

    2015-10-01

    The tumor microenvironment plays an important role in the progression of melanoma, the prototypical immunologic cutaneous malignancy. The triggering receptor expressed on myeloid cells (TREM) family of innate immune receptors modulates inflammatory and innate immune signaling. It has been investigated in various neoplastic diseases, but not in melanoma. This study examines the expression of TREM-1 (a proinflammatory amplifier) and TREM-2 (an anti-inflammatory modulator and phagocytic promoter) in human cutaneous melanoma and surrounding tissue. Indirect immunofluorescence staining was performed on skin biopsies from 10 melanoma patients and staining intensity was semiquantitatively scored. Expression of TREM-1 and TREM-2 was higher in keratinocytes than melanoma tissue (TREM-1: p < 0.01; TREM-2: p < 0.01). Whereas TREM-2 was the dominant isoform expressed in normal keratinocytes, TREM-1 expression predominated in melanoma tissue (TREM-1 to TREM-2 ratio: keratinocytes = 0.78; melanoma = 2.08; p < 0.01). The increased TREM ratio in melanoma tissue could give rise to a proinflammatory and protumor state of the microenvironment. This evidence may be suggestive of a TREM-1/TREM-2 paradigm in which relative levels dictate inflammatory and immune states, rather than absolute expression of one or the other. Further investigation regarding this paradigm is warranted and could carry prognostic or therapeutic value in treatment for melanoma. PMID:26184544

  20. Steroid receptor expression in the fish inner ear varies with sex, social status, and reproductive state

    PubMed Central

    2010-01-01

    Background Gonadal and stress-related steroid hormones are known to influence auditory function across vertebrates but the cellular and molecular mechanisms responsible for steroid-mediated auditory plasticity at the level of the inner ear remain unknown. The presence of steroid receptors in the ear suggests a direct pathway for hormones to act on the peripheral auditory system, but little is known about which receptors are expressed in the ear or whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA expression levels of multiple steroid receptor subtypes (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ; corticosteroid receptors: GR2, GR1a/b, MR) and aromatase in the main hearing organ of the inner ear (saccule) in the highly social African cichlid fish Astatotilapia burtoni, and tested whether these receptor levels were correlated with circulating steroid concentrations. Results We show that multiple steroid receptor subtypes are expressed within the main hearing organ of a single vertebrate species, and that expression levels differ between the sexes. We also show that steroid receptor subtype-specific changes in mRNA expression are associated with reproductive phase in females and social status in males. Sex-steroid receptor mRNA levels were negatively correlated with circulating estradiol and androgens in both males and females, suggesting possible ligand down-regulation of receptors in the inner ear. In contrast, saccular changes in corticosteroid receptor mRNA levels were not related to serum cortisol levels. Circulating steroid levels and receptor subtype mRNA levels were not as tightly correlated in males as compared to females, suggesting different regulatory mechanisms between sexes. Conclusions This is the most comprehensive study of sex-, social-, and reproductive-related steroid receptor mRNA expression in the peripheral auditory system of any single

  1. Determining TGF-β Receptor Levels in the Cell Membrane.

    PubMed

    Zhang, Long; Zhou, Fangfang; van Dinther, Maarten; Ten Dijke, Peter

    2016-01-01

    Transforming growth factor-β (TGF-β) is a pleiotropic cytokine that signals via transmembrane TGF-β type I and type II serine/threonine kinases receptors, i.e., TβRI and TβRII. Upon TGF-β-induced receptor complex formation, the TβRII kinase phosphorylates TβRI. Subsequently, the activated TβRI induces the phosphorylation of receptor regulated SMAD2 and SMAD3, which can form heteromeric complexes with Smad4. These heteromeric SMAD complexes accumulate in the nucleus, where they regulate target gene expression. The stability and membrane localization of TβRI is an important determinant to control the intensity and duration of TGF-β signaling. TβRI is targeted for poly-ubiquitylation-mediated proteasomal degradation by the SMAD7-SMURF E3 ligase complex. We recently identified another important regulatory factor that controls TβRI levels in the cell membrane. As a strong inducer of TGF-β signaling, ubiquitin-specific protease (USP) 4 was found to directly interact with TβRI and act as a deubiquitylating enzyme, thereby stabilizing TβRI levels at the plasma membrane. This chapter introduces methods for examining cell membrane receptor (TβRI) levels. PMID:26520116

  2. Sex Steroid Hormone Receptor Expression Affects Ovarian Cancer Survival12

    PubMed Central

    Jönsson, Jenny-Maria; Skovbjerg Arildsen, Nicolai; Malander, Susanne; Måsbäck, Anna; Hartman, Linda; Nilbert, Mef; Hedenfalk, Ingrid

    2015-01-01

    Background and Aims: Although most ovarian cancers express estrogen (ER), progesterone (PR), and androgen (AR) receptors, they are currently not applied in clinical decision making. We explored the prognostic impact of sex steroid hormone receptor protein and mRNA expression on survival in epithelial ovarian cancer. Methods: Immunohistochemical stainings for ERα, ERβ, PR, and AR were assessed in relation to survival in 118 serous and endometrioid ovarian cancers. Expression of the genes encoding the four receptors was studied in relation to prognosis in the molecular subtypes of ovarian cancer in an independent data set, hypothesizing that the expression levels and prognostic impact may differ between the subtypes. Results: Expression of PR or AR protein was associated with improved 5-year progression-free (P = .001 for both) and overall survival (P < .001 for both, log-rank test). ERα and ERβ did not provide prognostic information. Patients whose tumors coexpressed PR and AR had the most favorable prognosis, and this effect was retained in multivariable analyses. Analyses of the corresponding genes using an independent data set revealed differences among the molecular subtypes, but no clear relationship between high coexpression of PGR and AR and prognosis. Conclusions: A favorable outcome was seen for patients whose tumors coexpressed PR and AR. Gene expression data suggested variable effects in the different molecular subtypes. These findings demonstrate a prognostic role for PR and AR in ovarian cancer and support that tumors should be stratified based on molecular as well as histological subtypes in future studies investigating the role of endocrine treatment in ovarian cancer. PMID:26500033

  3. Molecular Background of Estrogen Receptor Gene Expression in Endometriotic Cells.

    PubMed

    Izawa, Masao; Taniguchi, Fuminori; Harada, Tasuku

    2016-07-01

    The molecular background of estrogen receptor (ER) expression is important to understand the pathophysiology of the high estrogen environment in endometriosis. However, the molecular details have not been fully understood. The objective of this study is to evaluate the molecular background of ERα and ERβ messenger RNA (mRNA) expression in endometriotic cells. The following summarizes our observations: (1) ERα mRNA expression in endometriotic cells was estimated to be approximately one-tenth of that in endometrial cells. (2) Three mRNAs, which include 3 different 5'-untranslated exons tagged to an open reading frame of wild-type ERα, were detected. (3) Expression of ERβ mRNA depends mostly on 0N promoter and includes 2 open reading frames: one for a wild-type ERβ1 and another for a splice variant ERβ2. (4) Expression of ERβ1 mRNA was approximately 40-fold higher than that in endometrial cells. (5) Expression of ERβ2 mRNA was almost at a comparable level of the ERβ1. 9 (6) ERα and ERβ mRNAs are equivalently expressed in endometriotic cells. These observations show the molecular background of ER mRNA expression in endometriotic cells and provide a clue to further understanding the estrogen-dependent pathophysiology leading to clinical application in endometriosis. PMID:26704524

  4. Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene.

    PubMed Central

    Kiely, J; Hadcock, J R; Bahouth, S W; Malbon, C C

    1994-01-01

    The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors. Images Figure 1 Figure 2 Figure 6 PMID:8092990

  5. Substance P receptor binding sites are expressed by glia in vivo after neuronal injury

    SciTech Connect

    Mantyh, P.W.; Johnson, D.J.; Boehmer, C.G.; Catton, M.D.; Vinters, H.V.; Maggio, J.E.; Too, Hengphon; Vigna, S.R. )

    1989-07-01

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, the authors examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

  6. Substance P Receptor Binding Sites are Expressed by Glia in vivo after Neuronal Injury

    NASA Astrophysics Data System (ADS)

    Mantyh, Patrick W.; Johnson, Donald J.; Boehmer, Christian G.; Catton, Mark D.; Vinters, Harry V.; Maggio, John E.; Too, Heng-Phon; Vigna, Steven R.

    1989-07-01

    In vitro studies have demonstrated that glia can express functional receptors for a variety of neurotransmitters. To determine whether similar neurotransmitter receptors are also expressed by glia in vivo, we examined the glial scar in the transected optic nerve of the albino rabbit by quantitative receptor autoradiography. Receptor binding sites for radiolabeled calcitonin gene-related peptide, cholecystokinin, galanin, glutamate, somatostatin, substance P, and vasoactive intestinal peptide were examined. Specific receptor binding sites for each of these neurotransmitters were identified in the rabbit forebrain but were not detected in the normal optic nerve or tract. In the transected optic nerve and tract, only receptor binding sites for substance P were expressed at detectable levels. The density of substance P receptor binding sites observed in this glial scar is among the highest observed in the rabbit forebrain. Ligand displacement and saturation experiments indicate that the substance P receptor binding site expressed by the glial scar has pharmacological characteristics similar to those of substance P receptors in the rabbit striatum, rat brain, and rat and canine gut. The present study demonstrates that glial cells in vivo express high concentrations of substance P receptor binding sites after transection of retinal ganglion cell axons. Because substance P has been shown to regulate inflammatory and immune responses in peripheral tissues, substance P may also, by analogy, be involved in regulating the glial response to injury in the central nervous system.

  7. Comparative study of histamine H4 receptor expression in human dermal fibroblasts.

    PubMed

    Ikawa, Yoshiko; Shiba, Kayoko; Ohki, Emi; Mutoh, Nanami; Suzuki, Masahiko; Sato, Hiromi; Ueno, Koichi

    2008-10-01

    The histamine H4 receptor (H4R) is the newest receptor identified of four histamine receptors. Its expression in numerous immune and inflammatory organs has been implicated in relation to immune systems and allergic diseases. In the present study, we demonstrate the expression of H4R in human dermal fibroblasts and investigate changes in its expression level when stimulated by histamine, phorbol 12-myristate 13-acetate (PMA), lipopolysaccharides (LPS), dexamethasone and indomethacin. Histamine and PMA showed no effects on H4R expression. LPS and indomethacin up-regulated H4R mRNA expression, and 20 microM dexamethasone increased H4R protein levels. These results indicate a good prospective for this new receptor in the development of effective treatments of inflammatory diseases and pruritus or for the appropriate prevention of toxicities. PMID:18827451

  8. The peroxisome proliferator-activated receptor α agonist, AZD4619, induces alanine aminotransferase-1 gene and protein expression in human, but not in rat hepatocytes: Correlation with serum ALT levels.

    PubMed

    Thulin, Petra; Bamberg, Krister; Buler, Marcin; Dahl, Björn; Glinghammar, Björn

    2016-09-01

    Alanine aminotransferase (ALT) in serum is the standard biomarker for liver injury. We have previously described a clinical trial with a novel selective peroxisome proliferator-activated receptor α (PPARα) agonist (AZD4619), which unexpectedly caused increased serum levels of ALT in treated individuals without any other evidence of liver injury. We pinpointed a plausible mechanism through which AZD4619 could increase serum ALT levels; namely through the PPARα-specific activation of the human ALT1 gene at the transcriptional level. In the present study, we present data from the preceding rat toxicity study, demonstrating that AZD4619 had no effect on rat serum ALT activity levels, and further experiments were performed to elucidate the mechanisms responsible for this species-related difference. Our results revealed that AZD4619 increased ALT1 protein expression in a dose-dependent manner in human, but not in rat primary hepatocytes. Cloning of the human and rat ALT1 promoters into luciferase vectors confirmed that AZD4619 induced only the human, but not the rat ALT1 gene promoter in a dose-dependent manner. In PPARα-GAL4 reporter gene assays, AZD4619 was >100-fold more potent on the human vs. rat PPARα levels, explaining the differences in induction of the ALT1 gene between the species at the concentration range tested. These data demonstrate the usefulness of the human and rat ALT1 reporter gene assays for testing future drug candidates at the preclinical stage. In drug discovery projects, these assays elucidate whether elevations in ALT levels observed in vivo or in the clinic are due to metabolic effects rather than a toxic event in the liver. PMID:27430334

  9. Regulation of gene expression in ovarian cancer cells by luteinizing hormone receptor expression and activation

    PubMed Central

    2011-01-01

    Background Since a substantial percentage of ovarian cancers express gonadotropin receptors and are responsive to the relatively high concentrations of pituitary gonadotropins during the postmenopausal years, it has been suggested that receptor activation may contribute to the etiology and/or progression of the neoplasm. The goal of the present study was to develop a cell model to determine the impact of luteinizing hormone (LH) receptor (LHR) expression and LH-mediated LHR activation on gene expression and thus obtain insights into the mechanism of gonadotropin action on ovarian surface epithelial (OSE) carcinoma cells. Methods The human ovarian cancer cell line, SKOV-3, was stably transfected to express functional LHR and incubated with LH for various periods of time (0-20 hours). Transcriptomic profiling was performed on these cells to identify LHR expression/activation-dependent changes in gene expression levels and pathways by microarray and qRT-PCR analyses. Results Through comparative analysis on the LHR-transfected SKOV-3 cells exposed to LH, we observed the differential expression of 1,783 genes in response to LH treatment, among which five significant families were enriched, including those of growth factors, translation regulators, transporters, G-protein coupled receptors, and ligand-dependent nuclear receptors. The most highly induced early and intermediate responses were found to occupy a network impacting transcriptional regulation, cell growth, apoptosis, and multiple signaling transductions, giving indications of LH-induced apoptosis and cell growth inhibition through the significant changes in, for example, tumor necrosis factor, Jun and many others, supportive of the observed cell growth reduction in in vitro assays. However, other observations, e.g. the substantial up-regulation of the genes encoding the endothelin-1 subtype A receptor, stromal cell-derived factor 1, and insulin-like growth factor II, all of which are potential therapeutic

  10. Elevated Resistin Gene Expression in African American Estrogen and Progesterone Receptor Negative Breast Cancer

    PubMed Central

    Vallega, Karin A.; Liu, NingNing; Myers, Jennifer S.; Yu, Kaixian; Sang, Qing-Xiang Amy

    2016-01-01

    Introduction African American (AA) women diagnosed with breast cancer are more likely to have aggressive subtypes. Investigating differentially expressed genes between patient populations may help explain racial health disparities. Resistin, one such gene, is linked to inflammation, obesity, and breast cancer risk. Previous studies indicated that resistin expression is higher in serum and tissue of AA breast cancer patients compared to Caucasian American (CA) patients. However, resistin expression levels have not been compared between AA and CA patients in a stage- and subtype-specific context. Breast cancer prognosis and treatments vary by subtype. This work investigates differential resistin gene expression in human breast cancer tissues of specific stages, receptor subtypes, and menopause statuses in AA and CA women. Methods Differential gene expression analysis was performed using human breast cancer gene expression data from The Cancer Genome Atlas. We performed inter-race resistin gene expression level comparisons looking at receptor status and stage-specific data between AA and CA samples. DESeq was run to test for differentially expressed resistin values. Results Resistin RNA was higher in AA women overall, with highest values in receptor negative subtypes. Estrogen-, progesterone-, and human epidermal growth factor receptor 2- negative groups showed statistically significant elevated resistin levels in Stage I and II AA women compared to CA women. In inter-racial comparisons, AA women had significantly higher levels of resistin regardless of menopause status. In whole population comparisons, resistin expression was higher among Stage I and III estrogen receptor negative cases. In comparisons of molecular subtypes, resistin levels were significant higher in triple negative than in luminal A breast cancer. Conclusion Resistin gene expression levels were significantly higher in receptor negative subtypes, especially estrogen receptor negative cases in AA

  11. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection

    PubMed Central

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-01-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP-/- MEF cells, and quite substantially decreased in TRIF-/- MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP-/- MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  12. Toll-Like Receptor Gene Expression during Trichinella spiralis Infection.

    PubMed

    Kim, Sin; Park, Mi Kyung; Yu, Hak Sun

    2015-08-01

    In Trichinella spiralis infection, type 2 helper T (Th2) cell-related and regulatory T (Treg) cell-related immune responses are the most important immune events. In order to clarify which Toll-like receptors (TLRs) are closely associated with these responses, we analyzed the expression of mouse TLR genes in the small intestine and muscle tissue during T. spiralis infection. In addition, the expression of several chemokine- and cytokine-encoding genes, which are related to Th2 and Treg cell mediated immune responses, were analyzed in mouse embryonic fibroblasts (MEFs) isolated from myeloid differentiation factor 88 (MyD88)/TIR-associated proteins (TIRAP) and Toll receptor-associated activator of interferons (TRIF) adapter protein deficient and wild type (WT) mice. The results showed significantly increased TLR4 and TLR9 gene expression in the small intestine after 2 weeks of T. spiralis infection. In the muscle, TLR1, TLR2, TLR5, and TLR9 gene expression significantly increased after 4 weeks of infection. Only the expression of the TLR4 and TLR9 genes was significantly elevated in WT MEF cells after treatment with excretory-secretory (ES) proteins. Gene expression for Th2 chemokine genes were highly enhanced by ES proteins in WT MEF cells, while this elevation was slightly reduced in MyD88/TIRAP(-/-) MEF cells, and quite substantially decreased in TRIF(-/-) MEF cells. In contrast, IL-10 and TGF-β expression levels were not elevated in MyD88/TIRAP(-/-) MEF cells. In conclusion, we suggest that TLR4 and TLR9 might be closely linked to Th2 cell and Treg cell mediated immune responses, although additional data are needed to convincingly prove this observation. PMID:26323841

  13. An Expression Refinement Process Ensures Singular Odorant Receptor Gene Choice.

    PubMed

    Abdus-Saboor, Ishmail; Al Nufal, Mohammed J; Agha, Maha V; Ruinart de Brimont, Marion; Fleischmann, Alexander; Shykind, Benjamin M

    2016-04-25

    Odorant receptor (OR) gene choice in mammals is a paradigmatic example of monogenic and monoallelic transcriptional selection, in which each olfactory sensory neuron (OSN) chooses to express one OR allele from over 1,000 encoded in the genome [1-3]. This process, critical for generation of the circuit from nose to brain [4-6], is thought to occur in two steps: a slow initial phase that randomly activates a single OR allele, followed by a rapid feedback that halts subsequent expression [7-14]. Inherent in this model is a finite failure rate wherein multiple OR alleles may be activated prior to feedback suppression [15, 16]. Confronted with more than one receptor, the neuron would need to activate a refinement mechanism to eliminate multigenic OR expression and resolve unique neuronal identity [16], critical to the generation of the circuit from nose to olfactory bulb. Here we used a genetic approach in mice to reveal a new facet of OR regulation that corrects adventitious activation of multiple OR alleles, restoring monogenic OR expression and unique neuronal identity. Using the tetM71tg model system, in which the M71 OR is expressed in >95% of mature OSNs and potently suppresses the expression of the endogenous OR repertoire [10], we provide clear evidence of a post-selection refinement (PSR) process that winnows down the number of ORs. We further demonstrate that PSR efficiency is linked to OR expression level, suggesting an underlying competitive process and shedding light on OR gene switching and the fundamental mechanism of singular OR choice. PMID:27040780

  14. Treadmill exercise enhances NMDA receptor expression in schizophrenia mice

    PubMed Central

    Park, Joon-Ki; Lee, Sam-Jun; Kim, Tae-Won

    2014-01-01

    Schizophrenia is a serious psychiatric disorder with several symptoms including cognitive dysfunction. Although the causes of schizophrenia are still unclear, there is a strong suspicion that the abnormality in N-methyl-D-aspartate (NMDA) receptor may contribute to schizophrenia symptoms. In the present study, the effect of treadmill exercise on the NMDA receptor expression was evaluated using MK-801-induced schizophrenia mice. Immunohistochemistry for expressions of NMDA receptor tyrosine hydroxylase (TH) was conducted. Western blot for brain-derived neurotrophic factor (BDNF) was also performed. In the present results, the mice in the MK-801-treated group displayed reduced NMDA receptor expression. Enhanced TH expression and suppressed BDNF expression were also observed in the MK-801-treated mice. Treadmill exercise improved NMDA receptor expression in the MK-801-induced schizophrenia mice. Treadmill exercise also suppressed TH expression and enhanced BDNF expression in the MK-801-induced schizophrenia mice. The present study showed that down-regulation of NMDA receptor demonstrated schizophrenia-like parameters, meanwhile treadmill running improved schizophrenia-related parameters through enhancing NMDA receptor expression. PMID:24678500

  15. Expression of Estrogen Receptor Alpha and Beta is Decreased in Hypospadias

    PubMed Central

    Qiao, Liang; Rodriguez, Esequiel; Weiss, Dana A.; Ferretti, Max; Risbridger, Gail; Cunha, Gerald R.; Baskin, Laurence S.

    2012-01-01

    Purpose Estrogenic endocrine disruptors acting via estrogen receptors α and β have been implicated in the etiology of hypospadias. However, the expression and distribution of estrogen receptors α and β in normal and hypospadiac human foreskins is unknown. We characterized the location and expression of estrogen receptors α and β in normal and hypospadiac foreskins. Materials and Methods We prospectively collected excess foreskin from 35 patients undergoing hypospadias repair and 15 patients undergoing elective circumcision. Hypospadias was classified as severe in 18 patients and mild in 17 based on the ectopic position of the meatus. mRNA expression levels in estrogen receptors α and β were quantified using reverse transcriptase polymerase chain reaction. Receptor location was characterized by immunohistochemical analysis. Additionally immunohistochemical analysis was performed in 4 archived human fetal penises. Results Mean ± SD ages were similar for the circumcision (9.5 ± 3 months) and hypospadias repair groups (9 ± 3 months, p = 0.75). mRNA expression levels in estrogen receptors α and β were significantly decreased in hypospadiac foreskin cases compared to controls (p <0.001), while no statistically significant differences were seen between foreskins with severe and mild hypospadias. Estrogen receptor β immunostaining was strong in normal foreskin but weak in hypospadiac foreskin. Estrogen receptor β immunoreactivity was most intense in the stratum basale and stratum spinosum. Estrogen receptor α immunostaining was weak in normal and mild hypospadias foreskin, and undetectable in severe hypospadias. Fetal penises expressed strong estrogen receptor β immunopositivity in the urethral plate epithelium, corpus spongiosum, corpora cavernosa and penile skin, while estrogen receptor α immunostaining was not detected. Conclusions These data demonstrate a difference in estrogen receptor α and β expression and location in the foreskin of patients

  16. Adolescent female rats exhibiting activity-based anorexia express elevated levels of GABA(A) receptor α4 and δ subunits at the plasma membrane of hippocampal CA1 spines.

    PubMed

    Aoki, Chiye; Sabaliauskas, Nicole; Chowdhury, Tara; Min, Jung-Yun; Colacino, Anna Rita; Laurino, Kevin; Barbarich-Marsteller, Nicole C

    2012-05-01

    Activity-based anorexia (ABA) is an animal model for anorexia nervosa that has revealed genetic links to anxiety traits and neurochemical characteristics within the hypothalamus. However, few studies have used this animal model to investigate the biological basis for vulnerability of pubertal and adolescent females to ABA, even though the great majority of the anorexia nervosa cases are females exhibiting the first symptoms during puberty. GABAergic inhibition of the hippocampus strongly regulates anxiety as well as plasticity throughout life. We recently showed that the hippocampal CA1 of female mice undergo a dramatic change at puberty onset--from expressing virtually none of the nonsynaptic α4βδ GABA(A) receptors (GABARs) prepubertally to expressing these GABARs at ~7% of the CA1 dendritic spine membranes at puberty onset. Furthermore, we showed that this change underlies the enhanced modulation of anxiety, neuronal excitability, and NMDA receptor-dependent synaptic plasticity in the hippocampus by the stress neurosteroid, THP (3α-OH-5α[β]-pregnan-20-one or [allo]pregnanolone). Here, we used quantitative electron microscopy to determine whether ABA induction in female rats during adolescence also elevates the expression of α4 and δ subunits of α4βδ GABARs, as was observed at puberty onset for mice. Our analysis revealed that rats also exhibit a rise of α4 and δ subunits of α4βδ GABARs at puberty onset, in that these subunits are detectable at ~6% of the dendritic spine membranes of CA1 pyramidal cells at puberty onset (postnatal day 32-36; P32-36) but this drops to about 2% by P40-P44. The levels of α4 and δ subunits at the CA1 spines remained low following exposure of females to either of the two environmental factors needed to generate ABA--food restriction and access to a running wheel for 4 days--from P40 to P44. This pattern contrasted greatly from those of ABA animals, for which the two environmental factors were combined. Within the

  17. Expression of the Endocannabinoid Receptors in Human Fascial Tissue

    PubMed Central

    Fede, C.; Albertin, G.; Petrelli, L.; Sfriso, M.M.; Biz, C.; Caro, R. De; Stecco, C.

    2016-01-01

    Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation. PMID:27349320

  18. Expression of the endocannabinoid receptors in human fascial tissue.

    PubMed

    Fede, C; Albertin, G; Petrelli, L; Sfriso, M M; Biz, C; De Caro, R; Stecco, C

    2016-01-01

    Cannabinoid receptors have been localized in the central and peripheral nervous system as well as on cells of the immune system, but recent studies on animal tissue gave evidence for the presence of cannabinoid receptors in different types of tissues. Their presence was supposed also in myofascial tissue, suggesting that the endocannabinoid system may help resolve myofascial trigger points and relieve symptoms of fibromyalgia. However, until now the expression of CB1 (cannabinoid receptor 1) and CB2 (cannabinoid receptor 2) in fasciae has not yet been established. Small samples of fascia were collected from volunteers patients during orthopedic surgery. For each sample were done a cell isolation, immunohistochemical investigation (CB1 and CB2 antibodies) and real time RT-PCR to detect the expression of CB1 and CB2. Both cannabinoid receptors are expressed in human fascia and in human fascial fibroblasts culture cells, although to a lesser extent than the control gene. We can assume that the expression of mRNA and protein of CB1 and CB2 receptors in fascial tissue are concentrated into the fibroblasts. This is the first demonstration that the fibroblasts of the muscular fasciae express CB1 and CB2. The presence of these receptors could help to provide a description of cannabinoid receptors distribution and to better explain the role of fasciae as pain generator and the efficacy of some fascial treatments. Indeed the endocannabinoid receptors of fascial fibroblasts can contribute to modulate the fascial fibrosis and inflammation. PMID:27349320

  19. Molecular Cooperativity Governs Diverse and Monoallelic Olfactory Receptor Expression

    NASA Astrophysics Data System (ADS)

    Xing, Jianhua; Tian, Xiaojun; Zhang, Hang; Sannerud, Jens

    Multiple-objective optimization is common in biological systems. In the mammalian olfactory system, each sensory neuron stochastically expresses only one out of up to thousands of olfactory receptor (OR) gene alleles; at organism level the types of expressed ORs need to be maximized. The molecular mechanism of this Nobel-Prize winning puzzle remains unresolved after decades of extensive studies. Existing models focus only on monoallele activation, and cannot explain recent observations in mutants, especially the reduced global diversity of expressed ORs in G9a/GLP knockouts. In this work we integrated existing information on OR expression, and proposed an evolutionarily optimized three-layer regulation mechanism, which includes zonal segregation, epigenetic and enhancer competition coupled to a negative feedback loop. This model not only recapitulates monoallelic OR expression, but also elucidates how the olfactory system maximizes and maintains the diversity of OR expression. The model is validated by several experimental results, and particularly underscores cooperativity and synergy as a general design principle of multi-objective optimization in biology. The work is supported by the NIGMS/DMS Mathematical Biology program.

  20. Class A scavenger receptor promotes osteoclast differentiation via the enhanced expression of receptor activator of NF-{kappa}B (RANK)

    SciTech Connect

    Takemura, Kenichi; Sakashita, Naomi; Fujiwara, Yukio; Komohara, Yoshihiro; Lei, XiaoFeng; Ohnishi, Koji; Suzuki, Hiroshi; Kodama, Tatsuhiko; Mizuta, Hiroshi; Takeya, Motohiro

    2010-01-22

    Osteoclasts originate from bone marrow monocyte/macrophage lineage cells, and their differentiation depends on macrophage colony-stimulating factor (M-CSF) and receptor activator nuclear factor kappa B (RANK) ligand. Class A scavenger receptor (SR-A) is one of the principal functional molecules of macrophages, and its level of expression declines during osteoclast differentiation. To investigate the role of SR-A in osteoclastogenesis, we examined pathological changes in femoral bone and the expression levels of osteoclastogenesis-related molecules in SR-A{sup -/-} mice. The femoral osseous density of SR-A{sup -/-} mice was higher than that of SR-A{sup +/+} mice, and the number of multinucleated osteoclasts was significantly decreased. An in vitro differentiation assay revealed that the differentiation of multinucleated osteoclasts from bone marrow-derived progenitor cells is impaired in SR-A{sup -/-} mice. Elimination of SR-A did not alter the expression level of the M-CSF receptor, c-fms; however, the expression levels of RANK and RANK-related osteoclast-differentiation molecules such as nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1) and microphthalmia-associated transcription factor (MITF) significantly decreased. Furthermore, acetylated low-density lipoprotein (AcLDL), an SR-A ligand, significantly increased the expression level of RANK and MITF during osteoclast differentiation. These data indicate that SR-A promotes osteoclastogenesis via augmentation of the expression level of RANK and its related molecules.

  1. Antipsychotic treatment modulates glutamate transport and NMDA receptor expression.

    PubMed

    Zink, Mathias; Englisch, Susanne; Schmitt, Andrea

    2014-11-01

    Schizophrenia patients often suffer from treatment-resistant cognitive and negative symptoms, both of which are influenced by glutamate neurotransmission. Innovative therapeutic strategies such as agonists at metabotropic glutamate receptors or glycin reuptake inhibitors try to modulate the brain's glutamate network. Interactions of amino acids with monoamines have been described on several levels, and first- and second-generation antipsychotic agents (FGAs, SGAs) are known to exert modulatory effects on the glutamatergic system. This review summarizes the current knowledge on effects of FGAs and SGAs on glutamate transport and receptor expression derived from pharmacological studies. Such studies serve as a control for molecular findings in schizophrenia brain tissue and are clinically relevant. Moreover, they may validate animal models for psychosis, foster basic research on antipsychotic substances and finally lead to a better understanding of how monoaminergic and amino acid neurotransmissions are intertwined. In the light of these results, important differences dependent on antipsychotic substances, dosage and duration of treatment became obvious. While some post-mortem findings might be confounded with multifold drug effects, others are unlikely to be influenced by antipsychotic treatment and could represent important markers of schizophrenia pathophysiology. In similarity to the convergence of toxic and psychotomimetic effects of dopaminergic, serotonergic and anti-glutamatergic substances, the therapeutic mechanisms of SGAs might merge on a yet to be defined molecular level. In particular, serotonergic effects of SGAs, such as an agonism at 5HT1A receptors, represent important targets for further clinical research. PMID:25214389

  2. Effects of chemotherapy agents on Sphingosine-1-Phosphate receptors expression in MCF-7 mammary cancer cells.

    PubMed

    Ghosal, P; Sukocheva, O A; Wang, T; Mayne, G C; Watson, D I; Hussey, D J

    2016-07-01

    Sphingosine-1-phosphate (S1P) is a potent bioactive sphingolipid involved in the regulation of cell proliferation and cancer progression. Increased expression of S1P receptors has been detected in advanced breast tumours with poor prognosis suggesting that S1P receptors might control tumour response to chemotherapy. However, it remains unclear how the levels of S1P receptor expression are influenced by chemotherapy agents. Western immunoblotting, PCR analysis and fluorescent microscopy techniques were used in this study to analyze expression patterns of S1P receptors 2 and 3 (S1P2/S1P3) in MCF-7 breast adenocarcinoma cells treated by Tamoxifen (TAM) and/or Medroxyprogesterone acetate (MPA). We found that TAM/MPA induce downregulation of S1P3 receptors, but stimulate expression of S1P2. According to cell viability and caspase activity analyses, as expected, TAM activated apoptosis. We also detected TAM/MPA-induced autophagy marked by formation of macroautophagosomes and increased level of Beclin 1. Combined application of TAM and MPA resulted in synergistic apoptosis- and autophagy-stimulating effects. Assessed by fluorescent microscopy with autophagosome marker LAMP-2, changes in S1P receptor expression coincided with activation of autophagy, suggestively, directing breast cancer cells towards death. Further studies are warranted to explore the utility of manipulation of S1P2 and S1P3 receptor expression as a novel treatment approach. PMID:27261597

  3. Sulfonylurea Receptor 1 Expression in Human Cerebral Infarcts

    PubMed Central

    Mehta, Rupal I.; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J.; Gerzanich, Volodymyr

    2013-01-01

    Abstract In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  4. Sulfonylurea receptor 1 expression in human cerebral infarcts.

    PubMed

    Mehta, Rupal I; Ivanova, Svetlana; Tosun, Cigdem; Castellani, Rudy J; Gerzanich, Volodymyr; Simard, J Marc

    2013-09-01

    In animal models of stroke, sulfonylurea receptor 1 (Sur1), a member of the adenosine triphosphate binding cassette transporter gene family, is transcriptionally upregulated in neural and vascular cells in which it plays a leading role in edema formation and necrotic cell death. To date, expression of Sur1 in the brains of humans with cerebral infarcts has not been systematically evaluated. We examined Sur1 expression in postmortem specimens obtained from 13 patients within the first 31 days after focal infarcts, 5 patients with lacunar infarcts, and 6 normal control brains using immunohistochemistry. Elevated immunoreactivity for Sur1 was detected in all cases of focal infarcts, with 3 distinct temporal patterns of expression: 1) neurons and endothelium showed the greatest elevation during the first week, after which levels declined; 2) astrocytes and microglia/macrophages showed progressive increases during the first 31 days; and 3) neutrophils near the infarct showed prominent immunoreactivity that did not change over time. Upregulation of Sur1 was corroborated using in situ hybridization for Abcc8 mRNA. Sulfonylurea receptor 1 immunoreactivity in lacunar infarcts was less prominent and more sporadic than in nonlacunar infarcts. In conjunction with previous studies, these data suggest that Sur1 may be a promising treatment target in patients with acute cerebral infarction. PMID:23965746

  5. Expression of prostanoid receptors in human lower segment pregnant myometrium.

    PubMed

    Leonhardt, Andreas; Glaser, Alexander; Wegmann, Markus; Hackenberg, Reinhard; Nüsing, Rolf M

    2003-11-01

    Prostanoids, especially prostaglandin (PG) E(2), are important mediators of uterine relaxation and contractions during gestation and parturition. Inhibitors of PG formation as well as PG analogues are used to modulate uterine tonus. So far, only limited data are available regarding the expression of prostanoid receptors in human pregnant myometrium. In the present study, the expression of the receptors for PGE(2) (EP1, EP2, EP3, EP4), PGF(2alpha) (FP), prostacyclin (IP), and thromboxane A(2) (TP) in human pregnant myometrium was studied by RT-PCR, in situ hybridization and immunohistochemistry. Myometrial tissue was obtained from five women at term and not in labour and from two women who delivered preterm. Tissue specimens were excised from the upper edge of the transverse lower uterine segment incision. In all tissues analysed, EP1, EP2, EP3, EP4, FP, TP and IP receptor mRNA and protein was detected. mRNA expression for PGD(2) (DP) receptor was not detected in the majority of tissue specimens. EP1, EP2, EP4, IP, TP and FP receptor protein was detected on myometrial smooth muscle cells, whereas EP3 receptor protein was only expressed by stromal and endothelial cells. In situ hybridization experiments yielded similar results. The expression of the EP2 receptor mRNA was inversely related to gestational age. We suggest that the contractile effect of PGE(2) at term is probably mediated directly by the EP1 receptor expressed in myometrial smooth muscle cells and indirectly by the EP3 receptor expressed in stromal cells and a decrease in EP2 receptor expression. PMID:14580364

  6. Melanocortin 4 Receptor and Dopamine D2 Receptor Expression in Brain Areas Involved in Food Intake

    PubMed Central

    Yoon, Ye Ran

    2015-01-01

    Background The melanocortin 4 receptor (MC4R) is involved in the regulation of homeostatic energy balance by the hypothalamus. Recent reports showed that MC4R can also control the motivation for food in association with a brain reward system, such as dopamine. We investigated the expression levels of MC4R and the dopamine D2 receptor (D2R), which is known to be related to food rewards, in both the hypothalamus and brain regions involved in food rewards. Methods We examined the expression levels of D2R and MC4R by dual immunofluorescence histochemistry in hypothalamic regions and in the bed nucleus of the stria terminalis (BNST), the central amygdala, and the ventral tegmental area of transgenic mice expressing enhanced green fluorescent protein under the control of the D2R gene. Results In the hypothalamic area, significant coexpression of MC4R and D2R was observed in the arcuate nucleus. We observed a significant coexpression of D2R and MC4R in the BNST, which has been suggested to be an important site for food reward. Conclusion We suggest that MC4R and D2R function in the hypothalamus for control of energy homeostasis and that within the brain regions related with rewards, such as the BNST, the melanocortin system works synergistically with dopamine for the integration of food motivation in the control of feeding behaviors. PMID:26790386

  7. 5-HT7 receptor activation promotes an increase in TrkB receptor expression and phosphorylation

    PubMed Central

    Samarajeewa, Anshula; Goldemann, Lolita; Vasefi, Maryam S.; Ahmed, Nawaz; Gondora, Nyasha; Khanderia, Chandni; Mielke, John G.; Beazely, Michael A.

    2014-01-01

    The serotonin (5-HT) type 7 receptor is expressed throughout the CNS including the cortex and hippocampus. We have previously demonstrated that the application of 5-HT7 receptor agonists to primary hippocampal neurons and SH-SY5Y cells increases platelet-derived growth factor (PDGF) receptor expression and promotes neuroprotection against N-methyl-D-aspartate-(NMDA)-induced toxicity. The tropomyosin-related kinase B (TrkB) receptor is one of the receptors for brain-derived neurotrophic factor (BDNF) and is associated with neurodevelopmental and neuroprotective effects. Application of LP 12 to primary cerebral cortical cultures, SH-SY5Y cells, as well as the retinal ganglion cell line, RGC-5, increased both the expression of full length TrkB as well as its basal phosphorylation state at tyrosine 816. The increase in TrkB expression and phosphorylation was observed as early as 30 min after 5-HT7 receptor activation. In addition to full-length TrkB, kinase domain-deficient forms may be expressed and act as dominant-negative proteins toward the full length receptor. We have identified distinct patterns of TrkB isoform expression across our cell lines and cortical cultures. Although TrkB receptor expression is regulated by cyclic AMP and Gαs-coupled GPCRs in several systems, we demonstrate that, depending on the model system, pathways downstream of both Gαs and Gα12 are involved in the regulation of TrkB expression by 5-HT7 receptors. Given the number of psychiatric and degenerative diseases associated with TrkB/BDNF deficiency and the current interest in developing 5-HT7 receptor ligands as pharmaceuticals, identifying signaling relationships between these two receptors will aid in our understanding of the potential therapeutic effects of 5-HT7 receptor ligands. PMID:25426041

  8. Scavenger receptor B1, the HDL receptor, is expressed abundantly in liver sinusoidal endothelial cells

    PubMed Central

    Ganesan, Latha P.; Mates, Jessica M.; Cheplowitz, Alana M.; Avila, Christina L.; Zimmerer, Jason M.; Yao, Zhili; Maiseyeu, Andrei; Rajaram, Murugesan V. S.; Robinson, John M.; Anderson, Clark L.

    2016-01-01

    Cholesterol from peripheral tissue, carried by HDL, is metabolized in the liver after uptake by the HDL receptor, SR-B1. Hepatocytes have long been considered the only liver cells expressing SR-B1; however, in this study we describe two disparate immunofluorescence (IF) experiments that suggest otherwise. Using high-resolution confocal microscopy employing ultrathin (120 nm) sections of mouse liver, improving z-axis resolution, we identified the liver sinusoidal endothelial cells (LSEC), marked by FcγRIIb, as the cell within the liver expressing abundant SR-B1. In contrast, the hepatocyte, identified with β-catenin, expressed considerably weaker levels, although optical resolution of SR-B1 was inadequate. Thus, we moved to a different IF strategy, first separating dissociated liver cells by gradient centrifugation into two portions, hepatocytes (parenchymal cells) and LSEC (non-parenchymal cells). Characterizing both portions for the cellular expression of SR-B1 by flow cytometry, we found that LSEC expressed considerable amounts of SR-B1 while in hepatocytes SR-B1 expression was barely perceptible. Assessing mRNA of SR-B1 by real time PCR we found messenger expression in LSEC to be about 5 times higher than in hepatocytes. PMID:26865459

  9. Androgen receptor transcriptionally regulates μ-opioid receptor expression in rat trigeminal ganglia.

    PubMed

    Lee, Ki Seok; Zhang, Youping; Asgar, Jamila; Auh, Q-Schick; Chung, Man-Kyo; Ro, Jin Y

    2016-09-01

    The involvement of testosterone in pain, inflammation, and analgesia has been reported, but the role of androgen receptor (AR), a steroid receptor for testosterone, is not well understood. We have previously shown that peripheral inflammation upregulates μ-opioid receptor (MOR) in rat trigeminal ganglia (TG) in a testosterone-dependent manner. In this study, we hypothesized that testosterone regulates MOR expression via transcriptional activities of AR in TG. We first examined whether AR is co-expressed with MOR in TG neurons. Our immunohistochemical experiment revealed that AR staining is detected in neurons of all sizes in TG and that a subset of AR is expressed in MOR as well as in TRPV1-positive neurons. We identified the promoter region of the rat MOR gene contains putative AR binding sites. Using chromatin immunoprecipitation assay, we demonstrated that AR directly binds to these sites in TG extracts. We confirmed with luciferase reporter assay that AR activated the MOR promoter in response to androgens in a human neuroblastoma cell line (5H-5YSY). These data demonstrated that AR functions as a transcriptional regulator of the MOR gene activity. Finally, we showed that flutamide, a specific AR antagonist, prevents complete Freund's adjuvant (CFA)-induced upregulation of MOR mRNA in TG, and that flutamide dose-dependently blocks the efficacy of DAMGO, a specific MOR agonist, on CFA-induced mechanical hypersensitivity. Our results expand the knowledge regarding the role of androgens and their receptor in pain and analgesia and have important clinical implications, particularly for inflammatory pain patients with low or compromised plasma testosterone levels. PMID:27320211

  10. Cannabinoid, melanocortin and opioid receptor expression on DRD1 and DRD2 subpopulations in rat striatum

    PubMed Central

    Oude Ophuis, Ralph J. A.; Boender, Arjen J.; van Rozen, Andrea J.; Adan, Roger A. H.

    2014-01-01

    The striatum harbors two neuronal populations that enable action selection. One population represents the striatonigral pathway, expresses the dopamine receptor D1 (DRD1) and promotes the execution of motor programs, while the other population represents the striatopallidal pathway, expresses the dopamine receptor D2 (DRD2) and suppresses voluntary activity. The two populations integrate distinct sensorimotor, cognitive, and emotional information streams and their combined activity enables the selection of adaptive behaviors. Characterization of these populations is critical to the understanding of their role in action selection, because it aids the identification of the molecular mechanisms that separate them. To that end, we used fluorescent in situ hybridization to quantify the percentage of striatal cells that (co)express dopaminergic receptors and receptors of the cannabinoid, melanocortin or opioid neurotransmitters systems. Our main findings are that the cannabinoid 1 receptor is equally expressed on both populations with a gradient from dorsal to ventral striatum, that the opioid receptors have a preference for expression with either the DRD1 or DRD2 and that the melanocortin 4 receptor (MC4R) is predominantly expressed in ventral parts of the striatum. In addition, we find that the level of MC4R expression determines its localization to either the DRD1 or the DRD2 population. Thereby, we provide insight into the sensitivity of the two dopaminoceptive populations to these neurotransmitters and progress the understanding of the mechanisms that enable action selection. PMID:24723856

  11. A second trigeminal CGRP receptor: function and expression of the AMY1 receptor

    PubMed Central

    Walker, Christopher S; Eftekhari, Sajedeh; Bower, Rebekah L; Wilderman, Andrea; Insel, Paul A; Edvinsson, Lars; Waldvogel, Henry J; Jamaluddin, Muhammad A; Russo, Andrew F; Hay, Debbie L

    2015-01-01

    Objective The trigeminovascular system plays a central role in migraine, a condition in need of new treatments. The neuropeptide, calcitonin gene-related peptide (CGRP), is proposed as causative in migraine and is the subject of intensive drug discovery efforts. This study explores the expression and functionality of two CGRP receptor candidates in the sensory trigeminal system. Methods Receptor expression was determined using Taqman G protein-coupled receptor arrays and immunohistochemistry in trigeminal ganglia (TG) and the spinal trigeminal complex of the brainstem in rat and human. Receptor pharmacology was quantified using sensitive signaling assays in primary rat TG neurons. Results mRNA and histological expression analysis in rat and human samples revealed the presence of two CGRP-responsive receptors (AMY1: calcitonin receptor/receptor activity-modifying protein 1 [RAMP1]) and the CGRP receptor (calcitonin receptor-like receptor/RAMP1). In support of this finding, quantification of agonist and antagonist potencies revealed a dual population of functional CGRP-responsive receptors in primary rat TG neurons. Interpretation The unexpected presence of a functional non-canonical CGRP receptor (AMY1) at neural sites important for craniofacial pain has important implications for targeting the CGRP axis in migraine. PMID:26125036

  12. Expression and function of P2 receptors in hematopoietic stem and progenitor cells

    PubMed Central

    Feng, Wenli; Wang, Lina

    2015-01-01

    Nucleotides have unambiguously emerged as a family of mediators of intercellular communication, which bind to a class of plasma membrane receptors, P2 receptors, to trigger intercellular signaling. P2 receptors can be further divided into P2X and P2Y subfamilies based on structure and function. Different hematopoietic cells express diverse spectrums of P2 receptors at different levels, including hematopoietic stem and progenitor cells (HSPCs). Extracellular adenosine triphosphate (ATP) exerts different effects on HSPCs, regulating cell proliferation, differentiation, migration, and chemotaxis, release of cytokines or lysosomal constituents, and generation of reactive oxygen or nitrogen species. The relationship between abnormal P2 receptor function and human diseases attracts more and more attention. This review summarizes the expression and function of P2 receptors in HSPCs and the relationship to hematopoietic diseases.

  13. Linking estrogen receptor β expression with inflammatory bowel disease activity

    PubMed Central

    Pierdominici, Marina; Maselli, Angela; Varano, Barbara; Barbati, Cristiana; Cesaro, Paola; Spada, Cristiano; Zullo, Angelo; Lorenzetti, Roberto; Rosati, Marco; Rainaldi, Gabriella; Limiti, Maria Rosaria; Guidi, Luisa

    2015-01-01

    Crohn disease (CD) and ulcerative colitis (UC) are chronic forms of inflammatory bowel disease (IBD) whose pathogenesis is only poorly understood. Estrogens have a complex role in inflammation and growing evidence suggests that these hormones may impact IBD pathogenesis. Here, we demonstrated a significant reduction (p < 0.05) of estrogen receptor (ER)β expression in peripheral blood T lymphocytes from CD/UC patients with active disease (n = 27) as compared to those in remission (n = 21) and healthy controls (n = 29). Accordingly, in a subgroup of CD/UC patients undergoing to anti-TNF-α therapy and responsive to treatment, ERβ expression was higher (p < 0.01) than that observed in not responsive patients and comparable to that of control subjects. Notably, ERβ expression was markedly decreased in colonic mucosa of CD/UC patients with active disease, reflecting the alterations observed in peripheral blood T cells. ERβ expression inversely correlated with interleukin (IL)-6 serum levels and exogenous exposure of both T lymphocytes and intestinal epithelial cells to this cytokine resulted in ERβ downregulation. These results demonstrate that the ER profile is altered in active IBD patients at both mucosal and systemic levels, at least in part due to IL-6 dysregulation, and highlight the potential exploitation of T cell-associated ERβ as a biomarker of endoscopic disease activity. PMID:26497217

  14. Identification of Neuropeptide Receptors Expressed by Melanin-Concentrating Hormone Neurons

    PubMed Central

    Parks, Gregory S.; Wang, Lien; Wang, Zhiwei; Civelli, Olivier

    2014-01-01

    Melanin-concentrating Hormone (MCH) is a 19 amino acid cyclic neuropeptide that acts in rodents via the MCH receptor 1 (MCHR1) to regulate a wide variety of physiological functions. MCH is produced by a distinct population of neurons located in the lateral hypothalamus (LH) and zona incerta (ZI) but MCHR1 mRNA is widely expressed throughout the brain. The physiological responses and behaviors regulated by the MCH system have been investigated, but less is known about how MCH neurons are regulated. The effects of most classical neurotransmitters on MCH neurons have been studied, but those of neuropeptides are poorly understood. In order to gain insight into how neuropeptides regulate the MCH system, we investigated which neuropeptide receptors are expressed by MCH neurons using double in situ hybridization. In all, twenty receptors, selected based upon either a suspected interaction with the MCH system or demonstrated high expression levels in the LH and ZI, were tested to determine whether they are expressed by MCH neurons. Overall, eleven neuropeptide receptors were found to exhibit significant colocalization with MCH neurons: Nociceptin / Orphanin FQ Opioid receptor (NOP), MCHR1, both Orexin receptors (ORX), Somatostatin receptor 1 and 2 (SSTR1, SSTR2), the Kisspeptin receotor (KissR1), Neurotensin receptor 1 (NTSR1), Neuropeptide S receptor (NPSR), Cholecystokinin receptor A (CCKAR) and the κ-opioid receptor (KOR). Of these receptors, six have never before been linked to the MCH system. Surprisingly, several receptors thought to regulate MCH neurons displayed minimal colocalization with MCH, suggesting that they may not directly regulate the MCH system. PMID:24978951

  15. Expression of functional leptin receptors in rodent Leydig cells.

    PubMed

    Caprio, M; Isidori, A M; Carta, A R; Moretti, C; Dufau, M L; Fabbri, A

    1999-11-01

    Several studies indicate that the size of body fat stores and the circulating levels of the adipocyte-derived hormone leptin are able to influence the activity of the hypothalamic-pituitary-gonadal axis. The leptin-hypothalamic-pituitary-gonadal interactions have been mainly studied at the level of the central nervous system. In this study, we investigated the possibility that leptin may have direct effects on the rodent Leydig cell function. To probe this hypothesis, we first analyzed the expression of leptin receptors (OB-R) in rodent Leydig cells in culture. RT-PCR studies showed that rat Leydig cells express both the long (OB-Rb) and short isoform (OB-Ra) of leptin receptor, whereas MLTC-1 cells (a murine Leydig tumor cell line) express only the long isoform. Short-term (30-90 min) incubation of rat Leydig cells with increasing concentrations ofleptin (2-500 ng/ml) led to a significant and dose-dependent inhibition of human (h)CG-stimulated testosterone (T) production (approximately 60% reduction, IC50 = 20 ng/ml) but no change in basal androgen release. Also, leptin (150 ng/ml) amplified hCG-induced intracellular cAMP formation (1- to 2-fold) without modifying basal cAMP levels. Subsequent experiments showed that leptin inhibited 8Br-cAMP-stimulated T production, indicating that leptin's effect is exerted beyond cAMP. The inhibitory effect of leptin on hCG-induced T secretion was accompanied by a significant reduction of androstenedione and a concomitant rise of the precursor metabolites pregnenolone, progesterone, and 17-OH-progesterone, conceivable with a leptin-induced lesion of 17,20 lyase activity. Separate experiments performed with the MLTC-1 cells (not expressing cytochrome P450-17alpha) showed that leptin, though amplifying hCG-stimulated cAMP production, did not modify hCG-stimulated pregnenolone and progesterone release. These results further indicate that leptin action on steroidogenesis occurs downstream of progesterone synthesis. Northern Blot

  16. Highly inducible expression from vectors containing multiple GRE's in CHO cells overexpressing the glucocorticoid receptor.

    PubMed Central

    Israel, D I; Kaufman, R J

    1989-01-01

    A conditional glucocorticoid-responsive expression vector system is described for highly inducible expression of heterologous genes in mammalian cells. This host-vector system requires high level expression of the glucocorticoid receptor (GR) protein in the host cell and multiple copies of the receptor binding site within the expression vector. Transfection and selection of Chinese hamster ovary cells with expression vectors encoding the rat GR yielded cell lines which express functional receptor at high levels. Insertion of multiple copies of the MMTV enhancer (glucocorticoid responsive element, GRE) into an Adenovirus major late promoter (AdMLP) based expression vector yielded greater than 1000-fold inducible expression by dexamethasone (dex) in transient DNA transfection assays. The induced expression level was 7-fold greater than that obtained with an AdMLP based vector containing an SV40 enhancer, but lacking GRE's. Vectors containing the SV40 enhancer in combination with multiple GRE's exhibited elevated basal expression in the absence of dex, but retained inducibility in both transient assays and after integration and amplification in the CHO genome. This expression system should be of general utility for studying gene regulation and for expressing heterologous genes in a regulatable fashion. Images PMID:2546123

  17. mRNA expression of dopamine receptors in peripheral blood lymphocytes of computer game addicts.

    PubMed

    Vousooghi, Nasim; Zarei, Seyed Zeinolabedin; Sadat-Shirazi, Mitra-Sadat; Eghbali, Fatemeh; Zarrindast, Mohammad Reza

    2015-10-01

    Excessive playing of computer games like some other behaviors could lead to addiction. Addictive behaviors may induce their reinforcing effects through stimulation of the brain dopaminergic mesolimbic pathway. The status of dopamine receptors in the brain may be parallel to their homologous receptors in peripheral blood lymphocytes (PBLs). Here, we have investigated the mRNA expression of dopamine D3, D4 and D5 receptors in PBLs of computer game addicts (n = 20) in comparison to normal subjects (n = 20), using a real-time PCR method. The results showed that the expression level of D3 and D4 dopamine receptors in computer game addicts were not statistically different from the control group. However, the expression of the mRNA of D5 dopamine receptor was significantly down-regulated in PBLs of computer game addicts and reached 0.42 the amount of the control group. It is concluded that unlike with drug addiction, the expression levels of the D3 and D4 dopamine receptors in computer game addicts are not altered compared to the control group. However, reduced level of the D5 dopamine receptor in computer game addicts may serve as a peripheral marker in studies where the confounding effects of abused drugs are unwanted. PMID:25967984

  18. Myoglobin expression in prostate cancer is correlated to androgen receptor expression and markers of tumor hypoxia.

    PubMed

    Meller, Sebastian; Bicker, Anne; Montani, Matteo; Ikenberg, Kristian; Rostamzadeh, Babak; Sailer, Verena; Wild, Peter; Dietrich, Dimo; Uhl, Barbara; Sulser, Tullio; Moch, Holger; Gorr, Thomas A; Stephan, Carsten; Jung, Klaus; Hankeln, Thomas; Kristiansen, Glen

    2014-10-01

    Recent studies identified unexpected expression and transcriptional complexity of the hemoprotein myoglobin (MB) in human breast cancer but its role in prostate cancer is still unclear. Expression of MB was immunohistochemically analyzed in three independent cohorts of radical prostatectomy specimens (n = 409, n = 625, and n = 237). MB expression data were correlated with clinicopathological parameters and molecular parameters of androgen and hypoxia signaling. Expression levels of novel tumor-associated MB transcript variants and the VEGF gene as a hypoxia marker were analyzed using qRT-PCR. Fifty-three percent of the prostate cancer cases were MB positive and significantly correlated with androgen receptor (AR) expression (p < 0.001). The positive correlation with CAIX (p < 0.001) and FASN (p = 0.008) as well as the paralleled increased expression of the tumor-associated MB transcript variants and VEGF suggest that hypoxia participates in MB expression regulation. Analogous to breast cancer, MB expression in prostate cancer is associated with steroid hormone signaling and markers of hypoxia. Further studies must elucidate the novel functional roles of MB in human carcinomas, which probably extend beyond its classic intramuscular function in oxygen storage. PMID:25172328

  19. Expression Profiles of Neuropeptides, Neurotransmitters, and Their Receptors in Human Keratocytes In Vitro and In Situ

    PubMed Central

    Słoniecka, Marta; Le Roux, Sandrine; Boman, Peter; Byström, Berit; Zhou, Qingjun; Danielson, Patrik

    2015-01-01

    Keratocytes, the quiescent cells of the corneal stroma, play a crucial role in corneal wound healing. Neuropeptides and neurotransmitters are usually associated with neuronal signaling, but have recently been shown to be produced also by non-neuronal cells and to be involved in many cellular processes. The aim of this study was to assess the endogenous intracellular and secreted levels of the neuropeptides substance P (SP) and neurokinin A (NKA), and of the neurotransmitters acetylcholine (ACh), catecholamines (adrenaline, noradrenaline and dopamine), and glutamate, as well as the expression profiles of their receptors, in human primary keratocytes in vitro and in keratocytes of human corneal tissue sections in situ. Cultured keratocytes expressed genes encoding for SP and NKA, and for catecholamine and glutamate synthesizing enzymes, as well as genes for neuropeptide, adrenergic and ACh (muscarinic) receptors. Keratocytes in culture produced SP, NKA, catecholamines, ACh, and glutamate, and expressed neurokinin-1 and -2 receptors (NK-1R and NK-2R), dopamine receptor D2, muscarinic ACh receptors, and NDMAR1 glutamate receptor. Human corneal sections expressed SP, NKA, NK-1R, NK-2R, receptor D2, choline acetyl transferase (ChAT), M3, M4 and M5 muscarinic ACh receptors, glutamate, and NMDAR1, but not catecholamine synthesizing enzyme or the α1 and β2 adrenoreceptors, nor M1 receptor. In addition, expression profiles assumed significant differences between keratocytes from the peripheral cornea as compared to those from the central cornea, as well as differences between keratocytes cultured under various serum concentrations. In conclusion, human keratocytes express an array of neuropeptides and neurotransmitters. The cells furthermore express receptors for neuropeptides/neurotransmitters, which suggests that they are susceptible to stimulation by these substances in the cornea, whether of neuronal or non-neuronal origin. As it has been shown that neuropeptides

  20. Expression of the human ABCC6 gene is induced by retinoids through the retinoid X receptor

    SciTech Connect

    Ratajewski, Marcin; Bartosz, Grzegorz; Pulaski, Lukasz . E-mail: lpulaski@cbm.pan.pl

    2006-12-01

    Mutations in the human ABCC6 gene are responsible for the disease pseudoxanthoma elasticum, although Physiological function or substrate of the gene product (an ABC transporter known also as MRP6) is not known. We found that the expression of this gene in cells of hepatic origin (where this gene is predominantly expressed in the body) is significantly upregulated by retinoids, acting as agonists of the retinoid X receptor (RXR) rather than the retinoid A receptor (RAR). The direct involvement of this nuclear receptor in the transcriptional regulation of ABCC6 gene expression was confirmed by transient transfection and chromatin immunoprecipitation assays. This constitutes the first direct proof of previously suggested involvement of nuclear hormone receptors in ABCC6 gene expression and the first identification of a transcription factor which may be relevant to regulation of ABCC6 level in tissues and in some PXE patients.

  1. Concerted Gene Expression of Hippocampal Steroid Receptors during Spatial Learning in Male Wistar Rats: A Correlation Analysis

    PubMed Central

    Lubec, Gert; Korz, Volker

    2016-01-01

    Adrenal and gonadal steroid receptor activities are significantly involved and interact in the regulation of learning, memory and stress. Thus, a coordinated expression of steroid receptor genes during a learning task can be expected. Although coexpression of steroid receptors in response to behavioral tasks has been reported the correlative connection is unclear. According to the inverted U-shape model of the impact of stress upon learning and memory we hypothesized that glucocorticoid (GR) receptor expression should be correlated to corticosterone levels in a linear or higher order manner. Other cognition modulating steroid receptors like estrogen receptors (ER) should be correlated to GR receptors in a quadratic manner, which describes a parabola and thus a U-shaped connection. Therefore, we performed a correlational meta-analyis of data of a previous study (Meyer and Korz, 2013a) of steroid receptor gene expressions during spatial learning, which provides a sufficient data basis in order to perform such correlational connections. In that study male rats of different ages were trained in a spatial holeboard or remained untrained and the hippocampal gene expression of different steroid receptors as well as serum corticosterone levels were measured. Expressions of mineralocorticoid (MR) and GR receptors were positively and linearly correlated with blood serum corticosterone levels in spatially trained but not in untrained animals. Training induced a cubic (best fit) relationship between mRNA levels of estrogen receptor α (ERα) and androgen receptor (AR) with MR mRNA. GR gene expression was linearly correlated with MR expression under both conditions. ERα m RNA levels were negatively and linearily and MR and GR gene expressions were cubicely correlated with reference memory errors (RME). Due to only three age classes correlations with age could not be performed. The findings support the U-shape theory of steroid receptor interaction, however the cubic fit

  2. Odor memories regulate olfactory receptor expression in the sensory periphery.

    PubMed

    Claudianos, Charles; Lim, Julianne; Young, Melanie; Yan, Shanzhi; Cristino, Alexandre S; Newcomb, Richard D; Gunasekaran, Nivetha; Reinhard, Judith

    2014-05-01

    Odor learning induces structural and functional modifications throughout the olfactory system, but it is currently unknown whether this plasticity extends to the olfactory receptors (Or) in the sensory periphery. Here, we demonstrate that odor learning induces plasticity in olfactory receptor expression in the honeybee, Apis mellifera. Using quantitative RT-PCR analysis, we show that six putative floral scent receptors were differentially expressed in the bee antennae depending on the scent environment that the bees experienced. Or151, which we characterized using an in vitro cell expression system as a broadly tuned receptor binding floral odorants such as linalool, and Or11, the specific receptor for the queen pheromone 9-oxo-decenoic acid, were significantly down-regulated after honeybees were conditioned with the respective odorants in an olfactory learning paradigm. Electroantennogram recordings showed that the neural response of the antenna was similarly reduced after odor learning. Long-term odor memory was essential for inducing these changes, suggesting that the molecular mechanisms involved in olfactory memory also regulate olfactory receptor expression. Our study demonstrates for the first time that olfactory receptor expression is experience-dependent and modulated by scent conditioning, providing novel insight into how molecular regulation at the periphery contributes to plasticity in the olfactory system. PMID:24628891

  3. Analysis of the cell surface expression of cytokine receptors using the surface protein biotinylation method.

    PubMed

    Pavel, Mahmud Arif; Lam, Clarissa; Kashyap, Parul; Salehi-Najafabadi, Zahra; Singh, Gurpreet; Yu, Yong

    2014-01-01

    Cytokines are pleiotropic, low-molecular-weight proteins that regulate the immune responses to infection and inflammation. They stimulate the immune responses by binding to cytokine receptors on the cell plasma membrane. Thus, knowledge of the expression level of particular cytokine receptors on cell surface is crucial for understanding the cytokine function and regulation. One of the techniques to explore the membrane embedded cytokine receptors is cell surface biotinylation. Biotinylated surface proteins can be rapidly purified through the strong interaction between biotin and streptavidin. Here, we describe the procedure for surface biotinylation and purification of biotinylated cytokine receptors for further downstream analysis. PMID:24908305

  4. Orphan nuclear receptor oestrogen-related receptor γ (ERRγ) plays a key role in hepatic cannabinoid receptor type 1-mediated induction of CYP7A1 gene expression.

    PubMed

    Zhang, Yaochen; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Jeong, Won-Il; Kim, Seong Heon; Lee, In-Kyu; Lee, Chul-Ho; Chiang, John Y L; Choi, Hueng-Sik

    2015-09-01

    Bile acids are primarily synthesized from cholesterol in the liver and have important roles in dietary lipid absorption and cholesterol homoeostasis. Detailed roles of the orphan nuclear receptors regulating cholesterol 7α-hydroxylase (CYP7A1), the rate-limiting enzyme in bile acid synthesis, have not yet been fully elucidated. In the present study, we report that oestrogen-related receptor γ (ERRγ) is a novel transcriptional regulator of CYP7A1 expression. Activation of cannabinoid receptor type 1 (CB1 receptor) signalling induced ERRγ-mediated transcription of the CYP7A1 gene. Overexpression of ERRγ increased CYP7A1 expression in vitro and in vivo, whereas knockdown of ERRγ attenuated CYP7A1 expression. Deletion analysis of the CYP7A1 gene promoter and a ChIP assay revealed an ERRγ-binding site on the CYP7A1 gene promoter. Small heterodimer partner (SHP) inhibited the transcriptional activity of ERRγ and thus regulated CYP7A1 expression. Overexpression of ERRγ led to increased bile acid levels, whereas an inverse agonist of ERRγ, GSK5182, reduced CYP7A1 expression and bile acid synthesis. Finally, GSK5182 significantly reduced hepatic CB1 receptor-mediated induction of CYP7A1 expression and bile acid synthesis in alcohol-treated mice. These results provide the molecular mechanism linking ERRγ and bile acid metabolism. PMID:26348907

  5. Expression, binding, and signaling properties of CRF2(a) receptors endogenously expressed in human retinoblastoma Y79 cells: passage-dependent regulation of functional receptors.

    PubMed

    Gutknecht, Eric; Hauger, Richard L; Van der Linden, Ilse; Vauquelin, Georges; Dautzenberg, Frank M

    2008-02-01

    Endogenous expression of the corticotropin-releasing factor type 2a receptor [CRF2(a)] but not CRF2(b) and CRF2(c) was observed in higher passage cultures of human Y79 retinoblastoma cells. Functional studies further demonstrated an increase in CRF2(a) mRNA and protein levels with higher passage numbers (> 20 passages). Although the CRF1 receptor was expressed at higher levels than the CRF2(a) receptor, both receptors were easily distinguishable from one another by selective receptor ligands. CRF(1)-preferring or non-selective agonists such as CRF, urocortin 1 (UCN1), and sauvagine stimulated cAMP production in Y79 to maximal responses of approximately 100 pmoles/10(5) cells, whereas the exclusive CRF2 receptor-selective agonists UCN2 and 3 stimulated cAMP production to maximal responses of approximately 25-30 pmoles/10(5) cells. UCN2 and 3-mediated cAMP stimulation was potently blocked by the approximately 300-fold selective CRF2 antagonist antisauvagine (IC50 = 6.5 +/- 1.6 nmol/L), whereas the CRF(1)-selective antagonist NBI27914 only blocked cAMP responses at concentrations > 10 microL. When the CRF(1)-preferring agonist ovine CRF was used to activate cAMP signaling, NBI27914 (IC50 = 38.4 +/- 3.6 nmol/L) was a more potent inhibitor than antisauvagine (IC50 = 2.04 +/- 0.2 microL). Finally, UCN2 and 3 treatment potently and rapidly desensitized the CRF2 receptor responses in Y79 cells. These data demonstrate that Y79 cells express functional CRF1 and CRF2a receptors and that the CRF2(a) receptor protein is up-regulated during prolonged culture. PMID:17976162

  6. Expression of chemokine decoy receptors and their ligands at the porcine maternal-fetal interface.

    PubMed

    Wessels, Jocelyn M; Linton, Nicola F; van den Heuvel, Marianne J; Cnossen, Sonya A; Edwards, Andrew K; Croy, Barbara Anne; Tayade, Chandrakant

    2011-02-01

    Successful pregnancy requires coordinated maternal-fetal cross-talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30-40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub-category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non-signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal-fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest. PMID:20680026

  7. Genomic organization and expression of insulin receptors in grass carp, Ctenopharyngodon idellus.

    PubMed

    Cai, Wenjing; Liang, Xu-Fang; Yuan, Xiaochen; Li, Aixuan; He, Yuhui; He, Shan

    2016-01-01

    Insulin receptors have been demonstrated to be involved in embryogenesis, food intake regulation and glucose metabolism in several fish, while more researchis needed for further understanding. In this study, the complete coding sequence (CDS) of insulin receptor a (insra) gene and insulin receptor b (insrb) gene in grass carp were obtained, the CDS were 4068 bp and 4514 bp in length, encoding 1355 aa protein and 1351 aa protein. Both of insra and insrb in grass carp showed high amino acid identities with other fish. Insra and insrb genes were widely expressed in all tested tissues with an overlapping but distinct expressions. The high levels of insra mRNA were distributed in hindgut and heart tissues. The insrb gene showed the highest expression levels in liver and hindgut. We also proved that two forms of grass carp insulin receptors participate in the regulation of blood glucose and might act differently. Phylogenetic analysis confirmed that different isoforms of fish insulin receptors are derived from two distinct genes, which was inconsistent with the generation of mammalian insulin receptors. Synteny analyses of insulin receptor genes showed that genes surrounding the insulin receptor genes were conserved in fish. Arhgef18, PEX11G, humanC19orf45 genes were highly conserved among mammal species. However, no conserved synteny was observed among fish, mammals, avians and amphibians. PMID:26772721

  8. Giant vesicles functionally expressing membrane receptors for an insect pheromone.

    PubMed

    Hamada, Satoshi; Tabuchi, Masashi; Toyota, Taro; Sakurai, Takeshi; Hosoi, Tomohiro; Nomoto, Tomonori; Nakatani, Kei; Fujinami, Masanori; Kanzaki, Ryohei

    2014-03-18

    To date, biochemical approaches to membrane receptors have been limited to the following methods: knockout or overexpression of membrane receptors by gene introduction and genome engineering or extraction of membrane receptor-surfactant complexes from innate cells and their introduction into model biomembranes. Here, we describe the development of a third method involving gene expression using cell-free in situ protein synthesis inside model biomembrane capsules. We verified this method by synthesizing olfactory receptors from the silkmoth Bombyx mori inside giant vesicles and found that they were excited in the presence of their ligand the Bombyx mori sex pheromone. PMID:24509495

  9. Mistargeting hippocampal axons by expression of a truncated Eph receptor

    PubMed Central

    Yue, Yong; Chen, Zhi-Yong; Gale, Nick W.; Blair-Flynn, Jan; Hu, Tian-Jing; Yue, Xin; Cooper, Margaret; Crockett, David P.; Yancopoulos, George D.; Tessarollo, Lino; Zhou, Renping

    2002-01-01

    Topographic mapping of axon terminals is a general principle of neural architecture that underlies the interconnections among many neural structures. The Eph family tyrosine kinase receptors and their ligands, the ephrins, have been implicated in the formation of topographic projection maps. We show that multiple Eph receptors and ligands are expressed in the hippocampus and its major subcortical projection target, the lateral septum, and that expression of a truncated Eph receptor in the mouse brain results in a pronounced alteration of the hippocamposeptal topographic map. Our observations provide strong support for a critical role of Eph family guidance factors in regulating ontogeny of hippocampal projections. PMID:12124402

  10. A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces.

    PubMed

    Tang, Yanjuan; Zhang, Shaolian; Wen, Qingqing; Huang, Hongxing; Yang, Peihui

    2015-06-30

    A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4×10(6)cellsmL(-1) with a detection limit of 40cellsmL(-1) was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35×10(5) with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening. PMID:26041531

  11. Identification and Expression Analysis of Putative Chemosensory Receptor Genes in Microplitis mediator by Antennal Transcriptome Screening

    PubMed Central

    Wang, Shan-Ning; Peng, Yong; Lu, Zi-Yun; Dhiloo, Khalid Hussain; Gu, Shao-Hua; Li, Rui-Jun; Zhou, Jing-Jiang; Zhang, Yong-Jun; Guo, Yu-Yuan

    2015-01-01

    Host-seeking, ovipositional behavior and mating of insects are controlled mainly by odor perception through sensory organs such as antennae. Antennal chemoreception is extremely important for insect survival. Several antennal chemosensory receptors are involved in mediating the odor detection in insects, especially the odorant receptors (ORs) and ionotropic receptors (IRs), to ensure the specificity of the olfactory sensory neuron responses. In the present study, we identified the chemosensory receptor gene repertoire of the parasitoid wasp Microplitis mediator, a generalist endoparasitoid that infests more than 40 types of Lepidopterous larvae and is widely distributed in the Palaearctic region. By transcriptome sequencing of male and female antennae we identified 60 candidate odorant receptors, six candidate ionotropic receptors and two gustatory receptors in M. mediator. The full-length sequences of these putative chemosensory receptor genes were obtained by using the rapid amplification of cDNA ends PCR (RACE-PCR) method. We also conducted reverse transcription PCR (RT-PCR) combined with real-time quantitative PCR (qPCR) for investigating the expression profiles of these chemosensory receptor genes in olfactory and non-olfactory tissues. The tissue- and sex-biased expression patterns may provide insights into the roles of the chemosensory receptor in M. mediator. Our findings support possible future study of the chemosensory behavior of M. mediator at the molecular level. PMID:26078716

  12. Nested expression domains for odorant receptors in zebrafish olfactory epithelium.

    PubMed

    Weth, F; Nadler, W; Korsching, S

    1996-11-12

    The mapping of high-dimensional olfactory stimuli onto the two-dimensional surface of the nasal sensory epithelium constitutes the first step in the neuronal encoding of olfactory input. We have used zebrafish as a model system to analyze the spatial distribution of odorant receptor molecules in the olfactory epithelium by quantitative in situ hybridization. To this end, we have cloned 10 very divergent zebrafish odorant receptor molecules by PCR. Individual genes are expressed in sparse olfactory receptor neurons. Analysis of the position of labeled cells in a simplified coordinate system revealed three concentric, albeit overlapping, expression domains for the four odorant receptors analyzed in detail. Such regionalized expression should result in a corresponding segregation of functional response properties. This might represent the first step of spatial encoding of olfactory input or be essential for the development of the olfactory system. PMID:8917589

  13. Effect of Ethylene Pathway Mutations upon Expression of the Ethylene Receptor ETR1 from Arabidopsis1

    PubMed Central

    Zhao, Xue-Chu; Qu, Xiang; Mathews, Dennis E.; Schaller, G. Eric

    2002-01-01

    The ethylene receptor family of Arabidopsis consists of five members, one of these being ETR1. The effect of ethylene pathway mutations upon expression of ETR1 was examined. For this purpose, ETR1 levels were quantified in mutant backgrounds containing receptor loss-of-function mutations, ethylene-insensitive mutations, and constitutive ethylene response mutations. Ethylene-insensitive mutations of ETR1 resulted in a posttranscriptional increase in levels of the mutant receptor. Treatment of seedlings with silver, which leads to ethylene insensitivity, also resulted in an increase in levels of ETR1. Loss-of-function mutations of ETR1 resulted in both transcriptional and posttranscriptional changes in levels of the receptor. Most other ethylene pathway mutations, including a newly isolated T-DNA insertion mutation in the gene encoding the ethylene receptor ERS1, had relatively minor effects upon the expression of ETR1. Our results indicate that mutations in ETR1 can affect expression at the posttranscriptional level, and suggest that these posttranscriptional changes may contribute to the phenotypes observed in the mutants. Our results also refine the model on how mutations in ethylene receptors are able to confer dominant ethylene insensitivity upon plants. PMID:12481081

  14. Dopamine Receptor Gene Expression in Human Amygdaloid Nuclei: Elevated D4 Receptor mRNAs in Major Depression

    PubMed Central

    Xiang, Lianbin; Szebeni, Katalin; Szebeni, Attila; Klimek, Violetta; Stockmeier, Craig A; Karolewicz, Beata; Kalbfleisch, John; Ordway, Gregory A

    2008-01-01

    Previous findings from this laboratory demonstrating changes in dopamine (DA) transporter and D2 receptors in the amygdaloid complex of subjects with major depression indicate that disruption of dopamine neurotransmission to the amygdala may contribute to behavioral symptoms associated with depression. Quantitative real-time RT-PCR was used to investigate the regional distribution of gene expression of DA receptors in the human amygdala. In addition, relative levels of mRNA of DA receptors in the basal amygdaloid nucleus were measured postmortem in subjects with major depression and normal control subjects. All five subtypes of DA receptor mRNA were detected in all amygdaloid subnuclei, although D1, D2, and D4 receptor mRNAs were more abundant than D3 and D5 mRNAs by an order of magnitude. The highest level of D1 mRNA was found in the central nucleus, whereas D2 mRNA was the most abundant in the basal nucleus. Levels of D4 mRNA were highest in the basal and central nuclei. In the basal nucleus, amounts of D4, but not D1 or D2, mRNAs were significantly higher in subjects with major depression and depressed suicide victims, as compared to control subjects. These findings demonstrate that the D1, D2 and D4 receptors are the major subtypes of DA receptors in the human amygdala. Elevated DA receptor gene expression in depressive subjects further implicates altered dopaminergic transmission in the amygdala in depression. PMID:18371940

  15. Serotonin 5-HT4 receptors and forebrain cholinergic system: receptor expression in identified cell populations.

    PubMed

    Peñas-Cazorla, Raúl; Vilaró, M Teresa

    2015-11-01

    Activation of serotonin 5-HT4 receptors has pro-cognitive effects on memory performance. The proposed underlying neurochemical mechanism is the enhancement of acetylcholine release in frontal cortex and hippocampus elicited by 5-HT4 agonists. Although 5-HT4 receptors are present in brain areas related to cognition, e.g., hippocampus and cortex, the cellular localization of the receptors that might modulate acetylcholine release is unknown at present. We have analyzed, using dual label in situ hybridization, the cellular localization of 5-HT4 receptor mRNA in identified neuronal populations of the rat basal forebrain, which is the source of the cholinergic innervation to cortex and hippocampus. 5-HT4 receptor mRNA was visualized with isotopically labeled oligonucleotide probes, whereas cholinergic, glutamatergic, GABAergic and parvalbumin-synthesizing neurons were identified with digoxigenin-labeled oligonucleotide probes. 5-HT4 receptor mRNA was not detected in the basal forebrain cholinergic cell population. In contrast, basal forebrain GABAergic, parvalbumin synthesizing, and glutamatergic cells contained 5-HT4 receptor mRNA. Hippocampal and cortical glutamatergic neurons also express this receptor. These results indicate that 5-HT4 receptors are not synthesized by cholinergic cells, and thus would be absent from cholinergic terminals. In contrast, several non-cholinergic cell populations within the basal forebrain and its target hippocampal and cortical areas express these receptors and are thus likely to mediate the enhancement of acetylcholine release elicited by 5-HT4 agonists. PMID:25183542

  16. Nuclear receptor SHP inhibition of Dnmt1 expression via ERRγ.

    PubMed

    Zhang, Yuxia; Wang, Li

    2011-05-01

    We describe a transcriptional mechanism regulating the expression of Dnmt1 by nuclear receptors. We show that ERRγ functions as a transcriptional activator of mouse and human Dnmt1 expression by direct binding to its response elements (ERE1/ERE2) in the dnmt1/DNMT1 promoters. The induction of Dnmt1 by ERRγ is repressed by SHP through SHP inhibition of ERRγ transactivity, diminishing ERRγ recruitment to the Dnmt1 promoter, and altering the conformation of local chromatin from an active mode by ERRγ to an inactive mode. Our study provides the first evidence for nuclear receptor mediated regulation of Dnmt1 expression through ERRγ and SHP crosstalk. PMID:21459093

  17. Nuclear receptor SHP inhibition of Dnmt1 expression via ERRγ

    PubMed Central

    Zhang, Yuxia; Wang, Li

    2011-01-01

    We describe a transcriptional mechanism regulating the expression of Dnmt1 by nuclear receptors. We show that ERRγ functions as a transcriptional activator of mouse and human Dnmt1 expression by direct binding to its response elements (ERE1/ERE2) in the dnmt1/DNMT1 promoters. The induction of Dnmt1 by ERRγ is repressed by SHP through SHP inhibition of ERRγ transactivity, diminishing ERRγ recruitment to the Dnmt1 promoter, and altering the conformation of local chromatin from an active mode by ERRγ to an inactive mode. Our study provides the first evidence for nuclear receptor mediated regulation of Dnmt1 expression through ERRγ and SHP crosstalk. PMID:21459093

  18. Strong Expression of Chemokine Receptor CXCR4 by Renal Cell Carcinoma Correlates with Advanced Disease

    PubMed Central

    Wehler, Thomas C.; Graf, Claudine; Biesterfeld, Stefan; Brenner, Walburgis; Schadt, Jörg; Gockel, Ines; Berger, Martin R.; Thüroff, Joachim W.; Galle, Peter R.; Moehler, Markus; Schimanski, Carl C.

    2008-01-01

    Diverse chemokines and their receptors have been associated with tumor growth, tumor dissemination, and local immune escape. In different tumor entities, the level of chemokine receptor CXCR4 expression has been linked with tumor progression and decreased survival. The aim of this study was to evaluate the influence of CXCR4 expression on the progression of human renal cell carcinoma. CXCR4 expression of renal cell carcinoma was assessed by immunohistochemistry in 113 patients. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human renal cell carcinoma revealed variable intensities of CXCR4 expression. Strong CXCR4 expression of renal cell carcinoma was significantly associated with advanced T-status (P = .039), tumor dedifferentiation (P = .0005), and low hemoglobin (P = .039). In summary, strong CXCR4 expression was significantly associated with advanced dedifferentiated renal cell carcinoma. PMID:19266088

  19. Differential expression of androgen, estrogen, and progesterone receptors in benign prostatic hyperplasia

    PubMed Central

    Song, Lingmin; Shen, Wenhao; Zhang, Heng; Wang, Qiwu; Wang, Yongquan; Zhou, Zhansong

    2016-01-01

    This study aimed to identify the differential expression levels of androgen receptor (AR), estrogen receptors (ERα, ERβ), and progesterone receptor (PGR) between normal prostate and benign prostatic hyperplasia (BPH). The combination of immunohistochemistry, quantitative real-time reverse transcription polymerase chain reaction, and Western blotting assay was used to identify the distribution and differential expression of these receptors at the immunoactive biomarker, transcriptional, and protein levels between 5 normal human prostate tissues and 40 BPH tissues. The results were then validated in a rat model of BPH induced by testosterone propionate and estradiol benzoate. In both human and rat prostate tissues, AR was localized mainly to epithelial and stromal cell nuclei; ERα was distributed mainly to stromal cells, but not exclusively; ERβ was interspersed in the basal layer of epithelium, but sporadically in epithelial and stromal cells; PGR was expressed abundantly in cytoplasm of epithelial and stromal cells. There were decreased expression of ERα and increased expression of PGR, but no difference in the expression of ERβ in the BPH compared to the normal prostate of both human and rat. Increased expression of AR in the BPH compared to the normal prostate of human was observed, however, the expression of AR in the rat prostate tissue was decreased. This study identified the activation of AR and PGR and repression of ERα in BPH, which indicate a promoting role of AR and PGR and an inhibitory role of ERα in the pathogenesis of BPH.

  20. Characterization of the Olfactory Receptors Expressed in Human Spermatozoa

    PubMed Central

    Flegel, Caroline; Vogel, Felix; Hofreuter, Adrian; Schreiner, Benjamin S. P.; Osthold, Sandra; Veitinger, Sophie; Becker, Christian; Brockmeyer, Norbert H.; Muschol, Michael; Wennemuth, Gunther; Altmüller, Janine; Hatt, Hanns; Gisselmann, Günter

    2016-01-01

    The detection of external cues is fundamental for human spermatozoa to locate the oocyte in the female reproductive tract. This task requires a specific chemoreceptor repertoire that is expressed on the surface of human spermatozoa, which is not fully identified to date. Olfactory receptors (ORs) are candidate molecules and have been attributed to be involved in sperm chemotaxis and chemokinesis, indicating an important role in mammalian spermatozoa. An increasing importance has been suggested for spermatozoal RNA, which led us to investigate the expression of all 387 OR genes. This study provides the first comprehensive analysis of OR transcripts in human spermatozoa of several individuals by RNA-Seq. We detected 91 different transcripts in the spermatozoa samples that could be aligned to annotated OR genes. Using stranded mRNA-Seq, we detected a class of these putative OR transcripts in an antisense orientation, indicating a different function, rather than coding for a functional OR protein. Nevertheless, we were able to detect OR proteins in various compartments of human spermatozoa, indicating distinct functions in human sperm. A panel of various OR ligands induced Ca2+ signals in human spermatozoa, which could be inhibited by mibefradil. This study indicates that a variety of ORs are expressed at the mRNA and protein level in human spermatozoa. PMID:26779489

  1. Aberrant expression of hormone receptors in adrenal Cushing's syndrome.

    PubMed

    Christopoulos, Stavroula; Bourdeau, Isabelle; Lacroix, André

    2004-01-01

    In recent years, a novel understanding of the pathophysiology of adrenal Cushing's syndrome has emerged. The ectopic or aberrant expression of G-protein-coupled hormone receptors in the adrenal cortex was found to play a central role in the regulation of cortisol secretion in ACTH-independent macronodular adrenal hyperplasia (AIMAH) and in some unilateral adrenal adenomas. Various aberrant receptors, functionally coupled to steroidogenesis, have been reported: GIP, vasopressin, beta-adrenergic, LH/hCG, and serotonin receptors have been best characterized, but angiotensin, leptin, glucagon, IL-1 and TSH receptors have also been described. The molecular mechanisms responsible for the aberrant expression of these receptors are currently unknown. One or many of these aberrant receptors are present in most cases of AIMAH and in some cases of adrenal adenomas with overt or sub-clinical secretion of cortisol. Clinical protocols to screen for such aberrant receptors have been developed and should be performed in all patients with AIMAH. The identification of such aberrant regulation of steroidogenesis in AIMAH provides the novel opportunity to treat some of these patients with pharmacological agents that either suppress the endogenous ligand or block the aberrant receptor, thus avoiding bilateral adrenalectomy. PMID:16010457

  2. Disrupted TSH Receptor Expression in Female Mouse Lung Fibroblasts Alters Subcellular IGF-1 Receptor Distribution.

    PubMed

    Atkins, Stephen J; Lentz, Stephen I; Fernando, Roshini; Smith, Terry J

    2015-12-01

    A relationship between the actions of TSH and IGF-1 was first recognized several decades ago. The close physical and functional associations between their respective receptors (TSHR and IGF-1R) has been described more recently in thyroid epithelium and human orbital fibroblasts as has the noncanonical behavior of IGF-1R. Here we report studies conducted in lung fibroblasts from female wild-type C57/B6 (TSHR(+/+)) mice and their littermates in which TSHR has been knocked out (TSHR(-/-)). Flow cytometric analysis revealed that cell surface IGF-1R levels are substantially lower in TSHR(-/-) fibroblasts compared with TSHR(+/+) fibroblasts. Confocal immunofluorescence microscopy revealed similar divergence with regard to both cytoplasmic and nuclear IGF-1R. Western blot analysis demonstrated both intact IGF-1R and receptor fragments in both cellular compartments. In contrast, IGF-1R mRNA levels were similar in fibroblasts from mice without and with intact TSHR expression. IGF-1 treatment of TSHR(+/+) fibroblasts resulted in reduced nuclear and cytoplasmic staining for IGF-1Rα, whereas it enhanced the nuclear signal in TSHR(-/-) cells. In contrast, IGF-1 enhanced cytoplasmic IGF-1Rβ in TSHR(-/-) fibroblasts while increasing the nuclear signal in TSHR(+/+) cells. These findings indicate the intimate relationship between TSHR and IGF-1R found earlier in human orbital fibroblasts also exists in mouse lung fibroblasts. Furthermore, the presence of TSHR in these fibroblasts influenced not only the levels of IGF-1R protein but also its subcellular distribution and response to IGF-1. They suggest that the mouse might serve as a suitable model for delineating the molecular mechanisms overarching these two receptors. PMID:26389690

  3. Expression of prostaglandin receptors in Chlamydia trachomatis-infected recurrent spontaneous aborters.

    PubMed

    Singh, Namita; Prasad, Priya; Singh, Laishram Chandreshwar; Das, Banashree; Rastogi, Sangita

    2016-06-01

    A study was undertaken to quantify the expression of prostaglandin (PG) receptors and find the effect of gestational age on expression of PG receptor genes in Chlamydia trachomatis-infected recurrent spontaneous aborters (RSA). Endometrial curettage tissue (ECT) was collected from 130 RSA (Group I) and 100 age-matched controls (Group II) at the Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi (India). PCR was performed for diagnosis of C. trachomatis cryptic plasmid; mRNA expression of PG receptor genes was assessed by real-time PCR (q-PCR), while serum progesterone/estrogen levels were determined by respective commercial kits. Data were evaluated statistically. A total of 15.4 % RSA (GroupI) were diagnosed as C. trachomatis-positive (200 bp), whereas controls were uninfected. q-PCR showed significant upregulation (P<0.0001) of PGE2 (EP-1, EP-2, EP-3, EP-4), PGF2α (FP) and PGI2 (IP) receptors in Group I versus Group II. The expression of PG receptors increased significantly with advanced gestational age (P<0.002); however, only contractile receptors, EP-1, EP-3 and FP, were positively correlated with gestational age in Group-I. In infected RSA, mean serum progesterone level was significantly low (P<0.0001) while serum oestrogen was high (P<0.0001). Overall, the data suggest that increased expression of PG receptors, particularly contractile gene receptors (EP-1, EP-3, FP), with advanced gestational age and altered steroid levels could be a possible risk factor for abortion in Chlamydia-infected RSA. PMID:27028620

  4. PKCε and allopregnanolone: functional cross-talk at the GABAA receptor level

    PubMed Central

    Puia, Giulia; Ravazzini, Federica; Castelnovo, Luca Franco; Magnaghi, Valerio

    2015-01-01

    Changes in GABAergic inhibition occur during physiological processes, during response to drugs and in various pathologies. These changes can be achieved through direct allosteric modifications at the γ-amino butyric acid (GABA) type A (GABAA) receptor protein level, or by altering the synthesis, trafficking and stability of the receptor. Neurosteroids (NSs) and protein kinase C (PKC) are potent modulators of GABAA receptors and their effects are presumably intermingled, even though evidence for this hypothesis is only partially explored. However, several PKC isoforms are able to phosphorylate the GABAA receptor, producing different functional effects. We focused on the ε isoform, that has been correlated to the sensitivity of the GABAA receptor to allosteric modulators and whose expression may be regulated in peripheral sensory neurons by NSs. The cross-talk between PKC-ε and NSs, leading to changes in GABAA receptor functionality, is considered and discussed in this perspective. PMID:25852476

  5. Endothelin ETA receptor expression in human cerebrovascular smooth muscle cells.

    PubMed Central

    Yu, J. C.; Pickard, J. D.; Davenport, A. P.

    1995-01-01

    1. Endothelin (ET) has been implicated in cerebrovasospasm for example, following subarachnoid haemorrhage, and blocking the interaction of ET with its receptors on cerebral vessels, may be of therapeutic benefit. The aim of our study was to characterize endothelin receptor sub-types on medial smooth muscle cells of human cerebral vessels. Cultures of vascular smooth muscle cells were explanted from human cerebral resistance vessels and characterized as human brain smooth muscle cells (HBSMCs). 2. Over a 48 h incubation period, HBSMC cultures secreted comparable levels of immunoreactive (IR) big endothelin-1 (big ET-1) and IR endothelin (ET): 12.7 +/- 10.3 and 8.3 +/- 5.6 pmol/10(6) cells, respectively (mean +/- s.e. mean from three different individuals), into the culture medium. 3. Total RNA was extracted from cultures of human brain smooth muscle cells. Reverse-transcriptase polymerase chain reaction (RI-PCR) assays and subsequent product separation by agarose gel electrophoresis revealed single bands corresponding to the expected product sizes encoding cDNA for ETA (299 base pairs) and ETB (428 base pairs) (n = 3 different cultures). 4. Autoradiography demonstrated the presence of specific binding sites for [125I]-ET-1 which labels all ET receptors, and [125I]-PD151242, an ETA subtype-selective antagonist which exclusively labels ETA receptors, but no specific-binding was detected using ETB subtype-selective [125I]-BQ3020 (n = 3 different cultures, in duplicate). 5. In saturation binding assays, [123I]-ET-1 bound with high affinity: KD = 0.8 +/- 0.1 nM and Bmax = 690 +/- 108 fmol mg-1. A one-site fit was preferred and Hill slopes were close to unity over the concentration range (10(-12) to 10(-8) M). [125I]-PD151242 also bound with similar affinity: KD = 0.4 +/- 0.1 nM and Bmax = 388 +/- 68 fmol mg-1 (mean +/- s.e. mean, n = 3 different cultures). Again, a one-site fit was preferred and Hill slopes were close to unity over the concentration range. Unlabelled PD

  6. Increased G Protein-Coupled Receptor Kinase (GRK) Expression in the Anterior Cingulate Cortex in Schizophrenia

    PubMed Central

    Funk, Adam J.; Haroutunian, Vahram; Meador-Woodruff, James H.; McCullumsmith, Robert E.

    2014-01-01

    Background Current pharmacological treatments for schizophrenia target G protein-coupled receptors (GPCRs), including dopamine receptors. Ligand bound GPCRs are regulated by a family of G protein-coupled receptor kinases (GRKs), members of which uncouple the receptor from heterotrimeric G proteins, desensitize the receptor, and induce receptor internalization via the arrestin family of scaffolding and signaling molecules. GRKs initiate the activation of downstream signaling pathways, can regulate receptors and signaling molecules independent of GPCR phosphorylation, and modulate epigenetic regulators like histone deacetylases (HDACs). We hypothesize that expression of GRK proteins are altered in schizophrenia, consistent with previous findings of alterations up and downstream from this family of molecules that facilitate intracellular signaling processes. Methods In this study we measured protein expression via Western blot analysis for GRKs 2, 3, 5, and 6 in the anterior cingulate cortex of patients with schizophrenia (N = 36) and a comparison group (N = 33). To control for antipsychotic treatment we measured these same targets in haloperidol treated vs. untreated rats (N = 10 for both). Results We found increased levels of GRK5 in schizophrenia. No changes were detected in GRK protein expression in rats treated with haloperidol decanoate for 9 months. Conclusion These data suggest that increased GRK5 expression may contribute the the pathophysiology of schizophrenia via abnormal regulation of the cytoskeleton, endocytosis, signaling, GPCRs, and histone modification. PMID:25153362

  7. Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications

    PubMed Central

    PONIEWIERSKA-BARAN, AGATA; SUSZYNSKA, MALWINA; SUN, WENYUE; ABDELBASET-ISMAIL, AHMED; SCHNEIDER, GABRIELA; BARR, FREDERIC G.; RATAJCZAK, MARIUSZ Z.

    2015-01-01

    The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression. PMID:26412593

  8. Human rhabdomyosarcoma cells express functional erythropoietin receptor: Potential therapeutic implications.

    PubMed

    Poniewierska-Baran, Agata; Suszynska, Malwina; Sun, Wenyue; Abdelbaset-Ismail, Ahmed; Schneider, Gabriela; Barr, Frederic G; Ratajczak, Mariusz Z

    2015-11-01

    The erythropoietin receptor (EpoR) is expressed by cells from the erythroid lineage; however, evidence has accumulated that it is also expressed by some solid tumors. This is an important observation, because recombinant erythropoietin (EPO) is employed in cancer patients to treat anemia related to chemo/radiotherapy. In our studies we employed eight rhabdomyosarcoma (RMS) cell lines (three alveolar-type RMS cell lines and five embrional-type RMS cell lines), and mRNA samples obtained from positive, PAX7-FOXO1-positive, and fusion-negative RMS patient samples. Expression of EpoR was evaluated by RT-PCR, gene array and FACS. The functionality of EpoR in RMS cell lines was evaluated by chemotaxis, adhesion, and direct cell proliferation assays. In some of the experiments, RMS cells were exposed to vincristine (VCR) in the presence or absence of EPO to test whether EPO may impair the therapeutic effect of VCR. We report for a first time that functional EpoR is expressed in human RMS cell lines as well as by primary tumors from RMS patients. Furthermore, EpoR is detectably expressed in both embryonal and alveolar RMS subtypes. At the functional level, several human RMS cell lines responded to EPO stimulation by enhanced proliferation, chemotaxis, cell adhesion, and phosphorylation of MAPKp42/44 and AKT. Moreover, RMS cells became more resistant to VCR treatment in the presence of EPO. Our findings have important potential clinical implications, indicating that EPO supplementation in RMS patients may have the unwanted side effect of tumor progression. PMID:26412593

  9. Coordinated regulation of natural killer receptor expression in the maturing human immune system

    PubMed Central

    Strauss-Albee, Dara M.; Horowitz, Amir; Parham, Peter; Blish, Catherine A.

    2014-01-01

    Natural killer (NK) cells are responsible for recognizing and killing transformed, stressed, and infected cells. They recognize a set of non-antigen-specific features termed “altered self” through combinatorial signals from activating and inhibitory receptors. These natural killer cell receptors (NKR) are also expressed on CD4+ and CD8+ T cells, B cells, and monocytes, though a comprehensive inventory of NKR expression patterns across leukocyte lineages has never been performed. Using mass cytometry, we found that NKR expression patterns distinguish cell lineages in human peripheral blood. In individuals with high levels of CD57, indicative of a mature immune repertoire, NKR are more likely to be expressed on non-NK cells, especially CD8+ T cells. Mature NK and CD8+ T cell populations show increased diversity of NKR surface expression patterns, but with distinct determinants: mature NK cells acquire primarily inhibitory receptors, while CD8+ T cells attain a specific subset of both activating and inhibitory receptors, potentially imbuing them with a distinct functional role. Concurrently, monocytes show decreased expression of the generalized inhibitory receptor LILRB1, consistent with an increased activation threshold. Therefore, NKR expression is coordinately regulated as the immune system matures, resulting in the transfer of “altered self” recognition potential among leukocyte lineages. This likely reduces antigen specificity in the mature human immune system, and implies that vaccines and therapeutics that engage both its innate and adaptive branches may be more effective in the settings of aging and chronic infection. PMID:25288567

  10. Reproductive Status Regulates Expression of Sex Steroid and GnRH Receptors in the Olfactory Bulb

    PubMed Central

    Fernald, Russell D.

    2010-01-01

    Neuromodulators including gonadotropin-releasing hormone (GnRH) and sex steroids help integrate an animal's internal physiological state with incoming external cues, and can have profound effects on the processing of behaviorally-relevant information, particularly from the olfactory system. While GnRH and steroid receptors are present in olfactory processing regions across vertebrates, little is known about whether their expression levels change with internal physiological state or external social cues. We used qRT-PCR to measure mRNA levels of two GnRH receptors (GnRH-R1, GnRH-R2), five sex steroid receptors (estrogen receptors: ERα, ERβa, ERβb; androgen receptors: ARα, ARβ), and aromatase in the olfactory bulb of the highly social African cichlid fish Astatotilapia burtoni. We asked whether these receptor levels changed with reproductive condition in females, or with social status, which regulates reproductive capacity in males. Our results reveal that mRNA levels of multiple sex steroid, GnRH receptor subtypes, and aromatase in the olfactory bulb vary with sex, social status in males, and reproductive condition in females, which highlights the potential importance of changing receptor levels in fine-tuning the olfactory system during the reproductive cycle. Further, steroid receptor mRNA levels were positively correlated with circulating steroid levels in males, but negatively correlated in females, suggesting different regulatory control between sexes. These results provide support for the hypothesis that the first-order olfactory relay station is a substrate for both GnRH and sex steroid modulation, and suggest that changes in receptor levels could be an important mechanism for regulating reproductive, social, and seasonal plasticity in olfactory perception observed across vertebrates. PMID:20466023

  11. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  12. Estrogen and Progesterone hormone receptor expression in oral cavity cancer

    PubMed Central

    Biegner, Thorsten; Teriete, Peter; Hoefert, Sebastian; Krimmel, Michael; Munz, Adelheid; Reinert, Siegmar

    2016-01-01

    Background Recent studies have shown an increase in the incidence of oral squamous cell carcinoma (OSCC) in younger patients. The hypothesis that tumors could be hormonally induced during pregnancy or in young female patients without the well-known risk factors alcohol or tobacco abuse seems to be plausible. Material and Methods Estrogen Receptor alpha (ERα) and Progesterone Receptor (PR) expression were analyzed in normal oral mucosa (n=5), oral precursor lesions (simple hyperplasia, n=11; squamous intraepithelial neoplasia, SIN I-III, n=35), and OSCC specimen. OSCCs were stratified in a young female (n=7) study cohort and older patients (n=46). In the young female study cohort three patients (n=3/7) developed OSCC during or shortly after pregnancy. Breast cancer tissues were used as positive control for ERα and PR expression. Results ERα expression was found in four oral precursor lesions (squamous intraepithelial neoplasia, SIN I-III, n=4/35, 11%) and in five OSCC specimen (n=5/46, 11%). The five ERα positive OSCC samples were older male patients. All patients within the young female study cohort were negatively stained for both ERα and PR. Conclusions ER expression could be regarded as a seldom risk factor for OSCC. PR expression seems to be not relevant for the development of OSCC. Key words:Oral squamous cell carcinoma, estrogen receptor, progesterone receptor, hormone receptor. PMID:27475696

  13. Regulation of vitamin D receptor expression by retinoic acid receptor alpha in acute myeloid leukemia cells.

    PubMed

    Marchwicka, Aleksandra; Cebrat, Małgorzata; Łaszkiewicz, Agnieszka; Śnieżewski, Łukasz; Brown, Geoffrey; Marcinkowska, Ewa

    2016-05-01

    Acute myeloid leukemia (AML) is the predominant acute leukemia among adults, characterized by an accumulation of malignant immature myeloid precursors. A very promising way to treat AML is differentiation therapy using either all-trans-retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D), or the use of both these differentiation-inducing agents. However, the effect of combination treatment varies in different AML cell lines, and this is due to ATRA either down- or up-regulating transcription of vitamin D receptor (VDR) in the cells examined. The mechanism of transcriptional regulation of VDR in response to ATRA has not been fully elucidated. Here, we show that the retinoic acid receptor α (RARα) is responsible for regulating VDR transcription in AML cells. We have shown that a VDR transcriptional variant, originating in exon 1a, is regulated by RARα agonists in AML cells. Moreover, in cells with a high basal level of RARα protein, the VDR gene is transcriptionally repressed as long as RARα agonist is absent. In these cells down-regulation of the level of RARα leads to increased expression of VDR. We consider that our findings provide a mechanistic background to explain the different outcomes from treating AML cell lines with a combination of ATRA and 1,25D. PMID:26969398

  14. β-Adrenergic receptor expression in vascular tumors.

    PubMed

    Chisholm, Karen M; Chang, Kay W; Truong, Mai T; Kwok, Shirley; West, Rob B; Heerema-McKenney, Amy E

    2012-11-01

    Propranolol has recently emerged as an effective therapy for infantile hemangiomas causing regression. The β-adrenergic receptor (AR) antagonist is thought to cause vasoconstriction by its effect on nitric oxide, block angiogenesis by its effect on vascular endothelial growth factor (VEGF), and induce apoptosis. In a prior report, we identified expression of β2-AR (B2-AR) and its phosphorylated form (B2-ARP) in a case of infantile hemangioma that responded to propranolol treatment. We now explore the expression of βARs on a variety of vascular lesions utilizing a tissue microarray containing 141 lesions, including infantile hemangiomas, angiosarcomas, hemangiomas, hemangioendotheliomas, and various vascular malformations. The array was immunostained for B2-AR, B2-ARP, and β3-AR (B3-AR), and the results scored for the intensity of endothelial cell expression as negative, weak positive, or strong positive. All phases of infantile hemangiomas had strong expression of all three receptors, with the exception of only weak expression of B2-ARP in the proliferative phase infantile hemangioma. Strong expression of all three receptors was present in many hemangiomas, hemangioendotheliomas, and vascular malformations. Absent to weak expression of all three receptors was seen in glomus tumor, hobnail hemangioendothelioma, pyogenic granuloma, and reactive vascular proliferations. This is the first study to report β-AR expression in a variety of vascular lesions. Although immunohistochemical expression of the receptors does not necessarily indicate that similar pathways of responsiveness to β-blockade are present, it does raises the possibility that β-blockade could potentially affect apoptosis and decrease responsiveness to VEGF. Additional study is warranted, as therapeutic options are limited for some patients with these lesions. PMID:22743651

  15. Construction of recombinant HEK293 cell lines for the expression of the neurotensin receptor NTSR1.

    PubMed

    Xiao, Su; Shiloach, Joseph; Grisshammer, Reinhard

    2015-01-01

    G protein-coupled receptors (GPCRs) are associated with a wide array of diseases and are targets of most of the medicines sold worldwide. Despite their clinical importance, only 25 unique GPCR structures have been determined as of April 2014. The first step for structural studies is to establish the expression of correctly folded, functional receptors in recombinant host cells at quantities to allow subsequent purification and crystallization trials. Here we describe the T-REx™-inducible expression system to construct and select a stable HEK293 cell line for high-level expression of functional neurotensin receptor type I (NTSR1). We also present the protocols used for the adaptation of the cells into suspension culture, as well as the optimization of the induction parameters for NTSR1 expression, which led to 1 mg of purified NTSR1 per liter suspension culture in bioreactors. PMID:25563176

  16. Disuse atrophy, plasma corticosterone, and muscle glucocorticoid receptor levels

    NASA Technical Reports Server (NTRS)

    Steffen, J. M.; Musacchia, X. J.

    1987-01-01

    The effect of whole-body suspension on the time course and the extent of plasma corticosterone changes and the tissue sensitivity to glucocorticoids were investigated in rats subjected to seven days of whole-body suspension. Plasma corticosterone increased significantly on the first and the third days of suspension, but returned to control levels by day seven. Muscle glucocorticoid receptors exhibited a characteristic hormonal specificity (evaluated in competitive-displacement experiments). In controls, receptor site concentration in the slow-twitch soleus was comparable to that in the fast-twitch gastrocnemius and plantaris, but was significantly less than in the extensor; seven days of suspension resulted in significant differential effects on muscle receptor levels. The largest increase in receptor concentration was observed in the soleus in which it remained elevated after the receptor levels in other muscles returned to normal.

  17. Defining Breast Cancer Intrinsic Subtypes by Quantitative Receptor Expression

    PubMed Central

    Cheang, Maggie C.U.; Martin, Miguel; Nielsen, Torsten O.; Prat, Aleix; Voduc, David; Rodriguez-Lescure, Alvaro; Ruiz, Amparo; Chia, Stephen; Shepherd, Lois; Ruiz-Borrego, Manuel; Calvo, Lourdes; Alba, Emilio; Carrasco, Eva; Caballero, Rosalia; Tu, Dongsheng; Pritchard, Kathleen I.; Levine, Mark N.; Bramwell, Vivien H.; Parker, Joel; Bernard, Philip S.; Ellis, Matthew J.; Perou, Charles M.; Di Leo, Angelo

    2015-01-01

    Purpose. To determine intrinsic breast cancer subtypes represented within categories defined by quantitative hormone receptor (HR) and HER2 expression. Methods. We merged 1,557 cases from three randomized phase III trials into a single data set. These breast tumors were centrally reviewed in each trial for quantitative ER, PR, and HER2 expression by immunohistochemistry (IHC) stain and by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), with intrinsic subtyping by research-based PAM50 RT-qPCR assay. Results. Among 283 HER2-negative tumors with <1% HR expression by IHC, 207 (73%) were basal-like; other subtypes, particularly HER2-enriched (48, 17%), were present. Among the 1,298 HER2-negative tumors, borderline HR (1%–9% staining) was uncommon (n = 39), and these tumors were heterogeneous: 17 (44%) luminal A/B, 12 (31%) HER2-enriched, and only 7 (18%) basal-like. Including them in the definition of triple-negative breast cancer significantly diminished enrichment for basal-like cancer (p < .05). Among 106 HER2-positive tumors with <1% HR expression by IHC, the HER2-enriched subtype was the most frequent (87, 82%), whereas among 127 HER2-positive tumors with strong HR (>10%) expression, only 69 (54%) were HER2-enriched and 55 (43%) were luminal (39 luminal B, 16 luminal A). Quantitative HR expression by RT-qPCR gave similar results. Regardless of methodology, basal-like cases seldom expressed ER/ESR1 or PR/PGR and were associated with the lowest expression level of HER2/ERBB2 relative to other subtypes. Conclusion. Significant discordance remains between clinical assay-defined subsets and intrinsic subtype. For identifying basal-like breast cancer, the optimal HR IHC cut point was <1%, matching the American Society of Clinical Oncology and College of American Pathologists guidelines. Tumors with borderline HR staining are molecularly diverse and may require additional assays to clarify underlying biology. PMID:25908555

  18. Regulation of pyruvate dehydrogenase kinase expression by the farnesoid X receptor

    SciTech Connect

    Savkur, Rajesh S.; Bramlett, Kelli S.; Michael, Laura F.; Burris, Thomas P. . E-mail: burris_thomas_p@lilly.com

    2005-04-01

    The pyruvate dehydrogenase complex (PDC) functions as an important junction in intermediary metabolism by influencing the utilization of fat versus carbohydrate as a source of fuel. Activation of PDC is achieved by phosphatases, whereas, inactivation is catalyzed by pyruvate dehydrogenase kinases (PDKs). The expression of PDK4 is highly regulated by the glucocorticoid and peroxisome proliferator-activated receptors. We demonstrate that the farnesoid X receptor (FXR; NR1H4), which regulates a variety of genes involved in lipoprotein metabolism, also regulates the expression of PDK4. Treatment of rat hepatoma cells as well as human primary hepatocytes with FXR agonists stimulates the expression of PDK4 to levels comparable to those obtained with glucocorticoids. In addition, treatment of mice with an FXR agonist significantly increased hepatic PDK4 expression, while concomitantly decreasing plasma triglyceride levels. Thus, activation of FXR may suppress glycolysis and enhance oxidation of fatty acids via inactivation of the PDC by increasing PDK4 expression.

  19. Developmental changes in NMDA receptor expression in the platyfish brain

    NASA Technical Reports Server (NTRS)

    Flynn, K. M.; Schreibman, M. P.; Magliulo-Cepriano, L.

    1997-01-01

    We have examined the distribution of the N-methyl-D-aspartate (NMDA) receptor in the brain of a freshwater teleost using an antibody against the R1 subunit of the receptor (NMDAR1). The primary site of localization was the nucleus olfactoretinalis (NOR), a significant gonadotropin releasing hormone (GnRH)-containing brain nucleus. The number of cells expressing NMDAR1 in this nucleus was dependent upon developmental stage, with pubescent and mature animals displaying significantly more stained cells than immature and senescent animals. This is the first reported observation of age- and maturity-related NMDA receptor association with GnRH-containing brain areas.

  20. Dopamine receptor gene expression by enkephalin neurons in rat forebrain

    SciTech Connect

    Le Moine, C.; Normand, E.; Guitteny, A.F.; Fouque, B.; Teoule, R.; Bloch, B. )

    1990-01-01

    In situ hybridization experiments were performed with brain sections from normal, control and haloperidol-treated rats to identify and map the cells expressing the D2 dopamine receptor gene. D2 receptor mRNA was detected with radioactive or biotinylated oligonucleotide probes. D2 receptor mRNA was present in glandular cells of the pituitary intermediate lobe and in neurons of the substantia nigra, ventral tegmental area, and forebrain, especially in caudate putamen, nucleus accumbens, olfactory tubercle, and piriform cortex. Hybridization with D2 and preproenkephalin A probes in adjacent sections, as well as combined hybridization with the two probes in the same sections, demonstrated that all detectable enkephalin neurons in the striatum contained the D2 receptor mRNA. Large neurons in caudate putamen, which were unlabeled with the preproenkephalin A probe and which may have been cholinergic, also expressed the D2 receptor gene. Haloperidol treatment (14 or 21 days) provoked an increase in mRNA content for D2 receptor and preproenkephalin A in the striatum. This suggests that the increase in D2 receptor number observed after haloperidol treatment is due to increased activity of the D2 gene. These results indicate that in the striatum, the enkephalin neurons are direct targets for dopamine liberated from mesostriatal neurons.

  1. TAM Receptors in Leukemia: Expression, Signaling, and Therapeutic Implications

    PubMed Central

    Brandão, Luis; Migdall-Wilson, Justine; Eisenman, Kristen; Graham, Douglas K.

    2016-01-01

    In the past 30 years there has been remarkable progress in the treatment of leukemia and lymphoma. However, current treatments are largely ineffective against relapsed leukemia and, in the case of pediatric patients, are often associated with severe long-term toxicities. Thus, there continues to be a critical need for the development of effective biologically targeted therapies. The TAM family of receptor tyrosine kinases—Tyro3, Axl, and Mer—plays an important role in normal hematopoiesis, including natural killer cell maturation, macrophage function, and platelet activation and signaling. Furthermore, TAM receptor activation leads to upregulation of pro-survival and proliferation signaling pathways, and aberrant TAM receptor expression contributes to cancer development, including myeloid and lymphoid leukemia. This review summarizes the role of TAM receptors in leukemia. We outline TAM receptor expression patterns in different forms of leukemia, describe potential mechanisms leading to their overexpression, and delineate the signaling pathways downstream of receptor activation that have been implicated in leukemogenesis. Finally, we discuss the current research focused on inhibitors against these receptors in an effort to develop new therapeutic strategies for leukemia. PMID:22150307

  2. Multiple melanocortin receptors are expressed in bone cells

    NASA Technical Reports Server (NTRS)

    Zhong, Qing; Sridhar, Supriya; Ruan, Ling; Ding, Ke-Hong; Xie, Ding; Insogna, Karl; Kang, Baolin; Xu, Jianrui; Bollag, Roni J.; Isales, Carlos M.

    2005-01-01

    Melanocortin receptors belong to the seven transmembrane domain, G-protein coupled family of receptors. There are five members of this receptor family labeled MC1R-MC5R. These receptors are activated by fragments derived from a larger molecule, proopiomelanocortin (POMC) and include ACTH, alpha beta and gamma-MSH and beta-endorphin. Because of in vitro and in vivo data suggesting direct effects of these POMC molecules on bone and bone turnover, we examined bone and bone derived cells for the presence of the various members of the melanocortin receptor family. We report that the five known melanocortin receptors are expressed to varying degrees in osteoblast-like and osteoclastic cells. POMC fragments increased proliferation and expression of a variety of genes in osteoblastic cells. Furthermore, POMC mRNA was detected in osteoclastic cells. These data demonstrate that POMC-derived peptide hormones acting through high affinity melanocortin receptors have specific effects on bone cells. Thus, in addition to the indirect effects of POMC-derived hormones on bone turnover through their modulation of steroid hormone secretion, POMC fragments may have direct and specific effects on bone cell subpopulations.

  3. mu opiate receptor: cDNA cloning and expression.

    PubMed Central

    Wang, J B; Imai, Y; Eppler, C M; Gregor, P; Spivak, C E; Uhl, G R

    1993-01-01

    mu opiate receptors recognize morphine with high affinity. A 2.1-kb rat brain cDNA whose predicted translation product displays 63% identity with recently described delta and kappa opiate receptor sequences was identified through polymerase chain reaction and cDNA homology approaches. This cDNA recognizes a 10.5-kb mRNA that is expressed in thalamic neurons. COS-cell expression confers naloxonazine-, Na(+)-, and GTP-sensitive binding of mu but not delta or kappa opioid ligands. Expressing cells bind morphine, [D-Ala2,N-methyl-Phe4,glyol5]enkephalin (DAMGO), and [D-Ala2,D-Leu5]enkephalin (DADLE) with nanomolar or subnanomolar affinities, defining a mu opiate receptor that avidly recognizes analgesic and euphoric opiate drugs and opioid peptides. Images Fig. 1 Fig. 3 PMID:8234282

  4. Impact of Cell Type and Epitope Tagging on Heterologous Expression of G Protein-Coupled Receptor: A Systematic Study on Angiotensin Type II Receptor

    PubMed Central

    Jiang, Lili; Teng, Gladys M. K.; Chan, Elaine Y. M.; Au, Shannon W. N.; Wise, Helen; Lee, Susanna S. T.; Cheung, Wing-Tai

    2012-01-01

    Despite heterologous expression of epitope-tagged GPCR is widely adopted for functional characterization, there is lacking of systematic analysis of the impact of expression host and epitope tag on GPCR expression. Angiotensin type II (AT2) receptor displays agonist-dependent and -independent activities, coupling to a spectrum of signaling molecules. However, consensus has not been reached on the subcellular distributions, signaling cascades and receptor-mediated actions. To examine the contributions of host cell and epitope tag on receptor expression and activity, epitope-tagged AT2 receptor variants were transiently or stably expressed in HEK293, CHO-K1 and PC12 cells. The epitope-tagged AT2 receptor variants were detected both on the cell membrane and in the perinuclear region. In transiently transfected HEK293 cells, Myc-AT2 existed predominantly as monomer. Additionally, a ladder of ubiquitinated AT2 receptor proteins was detected. By contrast, stably expressed epitope-tagged AT2 receptor variants existed as both monomer and high molecular weight complexes, and the latter was enriched in cell surface. Glycosylation promoted cell surface expression of Myc-AT2 but had no effect on AT2-GFP in HEK293 cells. In cells that stably expressed Myc-AT2, serum starvation induced apoptosis in CHO-K1 cells but not in HEK293 or PC12 cells. Instead, HEK293 and PC12 cells stably expressing Myc-AT2 exhibited partial cell cycle arrest with cells accumulating at G1 and S phases, respectively. Taken together, these results suggest that expression levels, subcellular distributions and ligand-independent constitutive activities of AT2 receptor were cell type-dependent while posttranslational processing of nascent AT2 receptor protein was modulated by epitope tag and mode of expression. PMID:23056563

  5. Cloning and olfactory expression of progestin receptors in the Chinese black sleeper Bostrichthys sinensis.

    PubMed

    Zhang, Yu Ting; Liu, Dong Teng; Zhu, Yong; Chen, Shi Xi; Hong, Wan Shu

    2016-05-01

    Our previous studies suggested that 17α,20β-dihydroxy-4-pregnen-3-one (DHP), an oocyte maturation inducing progestin, also acts as a sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. The electro-olfactogram response to DHP increased during the breeding season. In the present study, we cloned the cDNAs of the nine progestin receptors (pgr, paqr5, 6, 7(a, b), 8, 9, pgrmc1, 2) from B. sinensis, analyzed their tissue distribution, and determined the expression in the olfactory rosette during the reproductive cycle in female and male fish. The deduced amino acid sequences of the nine progestin receptors share high sequence identities with those of other fish species and relatively lower homology with their mammalian counterparts, and phylogenetic analyses classified the nine B. sinensis progestin receptors into their respective progestin receptor groups. Tissue distribution of B. sinensis progestin receptors showed differential expression patterns, but all these nine genes were expressed in the olfactory rosette. Interestingly, paqr5 mRNA was found in the intermediate and basal parts of the olfactory epithelium but not in the central core using in situ hybridization, and its expression level was the highest in the olfactory rosette among the tissues examined. These results suggested Paqr5 may have an important role for transmitting progestin signaling in the olfactory system. The expression levels of paqr7a and paqr7b, pgr and pgrmc2 mRNA peaked around the mid meiotic stage, and that of paqr8 peaked at late meiotic stage in the olfactory rosette in males, while the olfactory expression of paqr5 decreased gradually as spermatogenesis progressed. In contrast, the expression of the progestin receptors did not change significantly during the development of the ovary in the olfactory rosette in females, except that of pgr. Interestingly, the changes of paqr8 expression in

  6. The expression of histamine H4 receptor mRNA in the skin and other tissues of normal dogs.

    PubMed

    Eisenschenk, Melissa N C; Torres, Sheila M F; Oliveira, Simone; Been, Clint S

    2011-10-01

    The histamine 4 (H(4)) receptor was first cloned and characterized in 2000 using the human H(3) receptor DNA sequence. The H(4) receptor has been shown to participate in various aspects of inflammation, such as chemotaxis, upregulation of adhesion molecule expression and modulation of cytokine secretion. The primary goal of this study was to determine whether H(4) receptor mRNA is expressed in normal canine skin by performing an RT-PCR. An additional goal was to determine the expression of this receptor in the colon, liver, spleen and kidney. Tissues were collected from five healthy, young-adult pit bull dogs. Samples were immediately placed in RNAlater(®) solution and stored at -20°C until processed. The amplified products in all skin samples in addition to the colon, liver, spleen and kidney (variable expression) had the expected size of 400-500 bp. The sequenced amplicons matched the National Center for Biotechnology Information published sequence for the canine H(4) receptor. The study results showed that canine normal skin expresses the H(4) receptor mRNA. Further studies using immunohistochemistry should be conducted to demonstrate the expression of the H(4) receptor at the protein level and to localize the expression of this receptor in the skin. PMID:21392139

  7. Acetylcholine receptor-inducing factor from chicken brain increases the level of mRNA encoding the receptor. alpha. subunit

    SciTech Connect

    Harris, D.A.; Falls, D.L.; Dill-Devor, R.M.; Fischbach, G.D. )

    1988-03-01

    A 42-kDa glycoprotein isolated from chicken brain, referred to as acetylcholine receptor-inducing activity (ARIA), that stimulates the rate of incorporation of acetylcholine receptors into the surface of chicken myotubes may play a role in the nerve-induced accumulation of receptors at developing neuromuscular synapses. Using nuclease-protection assays, the authors have found that ARIA causes a 2- to 16-fold increase in the level of mRNA encoding the {alpha} subunit of the receptor, with little or no change in the levels of {gamma}- and {delta}-subunit messengers. ARIA also increases the amount of a putative nuclear precursor of {alpha}-subunit mRNA, consistent with an activation of gene transcription. These results suggest that the concentration of {alpha} subunit may limit the rate of biosynthesis of the acetylcholine receptors in chicken myotubes. They also indicate that neuronal factors can regulate the expression of receptor subunit genes in a selective manner. Tetrodotoxin, 8-bromo-cAMP, and forskolin also increase the amount of {alpha}-subunit mRNA, with little change in the amount of {gamma}- and {delta}-subunit mRNAs. Unlike ARIA, however, these agents have little effect on the concentration of the {alpha}-subunit nuclear precursor.

  8. Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain

    PubMed Central

    de Oliveira-Carlos, Vanessa; Ganz, Julia; Hans, Stefan; Kaslin, Jan; Brand, Michael

    2013-01-01

    The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches. PMID:24039926

  9. Developmentally Regulated Expression of the Nerve Growth Factor Receptor Gene in the Periphery and Brain

    NASA Astrophysics Data System (ADS)

    Buck, C. R.; Martinez, Humberto J.; Black, Ira B.; Chao, Moses V.

    1987-05-01

    Nerve growth factor (NGF) regulates development and maintenance of function of peripheral sympathetic and sensory neurons. A potential role for the trophic factor in brain has been detected only recently. The ability of a cell to respond to NGF is due, in part, to expression of specific receptors on the cell surface. To study tissue-specific expression of the NGF receptor gene, we have used sensitive cRNA probes for detection of NGF receptor mRNA. Our studies indicate that the receptor gene is selectively and specifically expressed in sympathetic (superior cervical) and sensory (dorsal root) ganglia in the periphery, and by the septum-basal forebrain centrally, in the neonatal rat in vivo. Moreover, examination of tissues from neonatal and adult rats reveals a marked reduction in steady-state NGF receptor mRNA levels in sensory ganglia. In contrast, a 2- to 4-fold increase was observed in the basal forebrain and in the sympathetic ganglia over the same time period. Our observations suggest that NGF receptor mRNA expression is developmentally regulated in specific areas of the nervous system in a differential fashion.

  10. Differential Expression of Functional Fc-Receptors and Additional Immune Complex Receptors on Mouse Kidney Cells

    PubMed Central

    Suwanichkul, Adisak; Wenderfer, Scott E.

    2013-01-01

    The precise mechanisms by which circulating immune complexes accumulate in the kidney to form deposits in glomerulonephritis are not well understood. In particular, the role of resident cells within glomeruli of the kidney has been widely debated. Immune complexes have been shown to bind one glomerular cell type (mesangial cells) leading to functional responses such as pro-inflammatory cytokine production. To further assess the presence of functional immunoreceptors on resident glomerular cells, cultured mouse renal epithelial, endothelial, and mesangial cells were treated with heat-aggregated mouse IgG or preformed murine immune complexes. Mesangial and renal endothelial cells were found to bind IgG complexes, whereas glomerular epithelial cell binding was minimal. A blocking antibody for Fc-gamma receptors reduced binding to mesangial cells but not renal endothelial cells, suggesting differential immunoreceptor utilization. RT-PCR and immunostaining based screening of cultured renal endothelial cells showed limited low-level expression of known Fc-receptors and Igbinding proteins. The interaction between mesangial cells and renal endothelial cells and immune complexes resulted in distinct, cell-specific patterns of chemokine and cytokine production. This novel pathway involving renal endothelial cells likely contributes to the predilection of circulating immune complex accumulation within the kidney and to the inflammatory responses that drive kidney injury. PMID:23911392

  11. Differential effects of UV irradiation on nuclear retinoid receptor levels in cultured keratinocytes and melanocytes.

    PubMed

    Andersson, Eva; Rosdahl, Inger; Törmä, Hans; Vahlquist, Anders

    2003-10-01

    A major risk factor for skin cancer is UV irradiation, which not only damages DNA and other photosensitive compounds like vitamin A, but may also perturb cellular signaling, e.g. via the retinoid receptor system believed to be important for cancer protection. We used cultured normal human keratinocytes and melanocytes to examine the effects of UV irradiation on the expression of the predominant retinoid receptors in the human skin (RARalpha, RARgamma and RXRalpha) and the AP-1 protein c-Jun; mRNA levels were studied by real-time PCR and protein levels by Western blot. In keratinocytes, a single dose of UVB (50 mJ/cm2) caused a rapid drop in the expression of all three receptors (mRNA levels minus 35-50% after 4 h; protein levels minus 20-45% after 8 h), which was followed over the next 40 h by a variable response, leading to full normalization for RARalpha only. In contrast, the levels of c-Jun did not change significantly after UV exposure. In melanocytes, UVB caused a similar drop of the retinoid receptor levels as in keratinocytes but this was soon followed by an increased expression leading to a complete normalization of all receptor levels within 1-3 days. The c-Jun levels in melanocytes increased 1 day after UV exposure and remained high (plus 50%) thereafter. In both cell types, a approximately 3-fold increase in apoptosis (measured by DNA fragmentation) was observed 8-48 h after UVB irradiation. In conclusion, a depletion of vitamin A and retinoid receptors by UV irradiation, together with unchanged or even increased c-Jun levels, might seriously interfere with retinoid signaling and thus promote future tumor development, especially in keratinocytes. PMID:14705796

  12. Regulation of retinoid X receptor gamma expression by fed state in mouse liver

    SciTech Connect

    Park, Sangkyu; Lee, Yoo Jeong; Ko, Eun Hee; Kim, Jae-woo

    2015-02-27

    Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting–feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting–feeding cycle. - Highlights: • Refeeding increases the RXRγ expression level in mouse liver. • RXRγ expression is induced by high glucose condition in primary hepatocytes. • RXRγ and LXRα have synergistic effect on SREBP-1c promoter activity. • RXRγ binds to LXRE(-299/-280) located within SREBP-1c promoter region and interacts with LXRα.

  13. Sexual differences of imprinted genes' expression levels.

    PubMed

    Faisal, Mohammad; Kim, Hana; Kim, Joomyeong

    2014-01-01

    In mammals, genomic imprinting has evolved as a dosage-controlling mechanism for a subset of genes that play critical roles in their unusual reproduction scheme involving viviparity and placentation. As such, many imprinted genes are highly expressed in sex-specific reproductive organs. In the current study, we sought to test whether imprinted genes are differentially expressed between the two sexes. According to the results, the expression levels of the following genes differ between the two sexes of mice: Peg3, Zim1, Igf2, H19 and Zac1. The expression levels of these imprinted genes are usually greater in males than in females. This bias is most obvious in the developing brains of 14.5-dpc embryos, but also detected in the brains of postnatal-stage mice. However, this sexual bias is not obvious in 10.5-dpc embryos, a developmental stage before the sexual differentiation. Thus, the sexual bias observed in the imprinted genes is most likely attributable by gonadal hormones rather than by sex chromosome complement. Overall, the results indicate that several imprinted genes are sexually different in terms of their expression levels, and further suggest that the transcriptional regulation of these imprinted genes may be influenced by unknown mechanisms associated with sexual differentiation. PMID:24125951

  14. Expression of neurotrophins and their receptors in human CD34+ bone marrow cells.

    PubMed

    Paczkowska, E; Piecyk, K; Luczkowska, K; Kotowski, M; Roginska, D; Pius-Sadowska, E; Oronowicz, K; Ostrowski, M; Machalinski, B

    2016-02-01

    Bone marrow (BM) CD34+ cells have the ability to secrete growth factors, cytokines, and chemotactic factors. We sought to better characterize this population and to investigate whether human BM CD34+ cells express neurotrophins (NTs) and their relevant receptors. We also compared their expression levels with BM nucleated cells (NCs). BM CD34+ cells were evaluated with respect to the expression levels of neurotrophins using qRT-PCR, immunofluorescent staining, and Western blotting. Next, the expression of specific (TrkA, TrkB, TrkC) and non-specific (p75NTR) neurotrophin receptors was detected by qRT-PCR and immunofluorescent staining in BM CD34+ cells. Using qRT- PCR, we show that even in the absence of inducing factors, CD34+ cells spontaneously express neurotrophins such as NGF, BDNF, NT-3, and NT-4. In addition, the NT expression levels in BM CD34+ cells are considerably higher than in NCs. Furthermore, we confirmed intracellular NT expression in BM CD34+ cells at the protein level using immunofluorescent staining and Western blotting. Using qRT-PCR, we found that immunomagnetically separated BM CD34+ cells spontaneously express high-affinity neurotrophin receptors (TrkA, TrkB, and TrkC) and the low-affinity receptor p75NTR at higher levels than NCs. Immunomagnetic CD34+ cell separation enables for the rapid and gentle sorting of stem/progenitor cells (SPCs) to prepare specific cell types for use in research and clinical applications. Our study suggests that BM CD34+ cells have the potential to support trophic factors for neural tissue and could contribute towards the protection and regeneration of neural cells. PMID:27010904

  15. Nicotinic receptor Alpha7 expression during mouse adrenal gland development.

    PubMed

    Gahring, Lorise C; Myers, Elizabeth; Palumbos, Sierra; Rogers, Scott W

    2014-01-01

    The nicotinic acetylcholine receptor alpha 7 (α7) is a ligand-activated ion channel that contributes to a diversity of cellular processes involved in development, neurotransmission and inflammation. In this report the expression of α7 was examined in the mouse developing and adult adrenal gland that expresses a green fluorescent protein (GFP) reporter as a bi-cistronic extension of the endogenous α7 transcript (α7(G)). At embryonic day 12.5 (E12.5) α7(G) expression was associated with the suprarenal ganglion and precursor cells of the adrenal gland. The α7(G) cells are catecholaminergic chromaffin cells as reflected by their progressive increase in the co-expression of tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH) that is complete by E18.5. In the adult, α7(G) expression is limited to a subset of chromaffin cells in the adrenal medulla that cluster near the border with the adrenal cortex. These chromaffin cells co-express α7(G), TH and DBH, but they lack phenylethanolamine N-methyltransferase (PNMT) consistent with only norepinephrine (NE) synthesis. These cell groups appear to be preferentially innervated by pre-ganglionic afferents identified by the neurotrophin receptor p75. No afferents identified by beta-III tubulin, neurofilament proteins or p75 co-expressed α7(G). Occasional α7(G) cells in the pre-E14.5 embryos express neuronal markers consistent with intrinsic ganglion cells and in the adult some α7(G) cells co-express glutamic acid decarboxylase. The transient expression of α7 during adrenal gland development and its prominent co-expression by a subset of NE chromaffin cells in the adult suggests that the α7 receptor contributes to multiple aspects of adrenal gland development and function that persist into adulthood. PMID:25093893

  16. Expression of somatostatin receptor genes and acetylcholine receptor development in rat skeletal muscle during postnatal development.

    PubMed

    Peng, M; Conforti, L; Millhorn, D E

    1998-05-01

    Our laboratory reported previously that somatostatin (SST) is transiently expressed in rat motoneurons during the first 14 days after birth. We investigated the possibility that the SST receptor (SSTR) is expressed in skeletal muscle. We found that two of the five subtypes of SSTR (SSTR3 and SSTR4) are expressed in skeletal muscle with a time course that correlates with the transient expression of SST in motoneurons. In addition, SSTR2A is expressed from birth to adulthood in skeletal muscle. Both SSTR2A and SSTR4 are also expressed in L6 cells, a skeletal muscle cell line. Somatostatin acting through its receptors has been shown to stimulate tyrosine phosphatase activity in a number of different tissues. We found that several proteins (50, 65, 90, 140, 180 and 200 kDa) exhibited a reduced degree of tyrosine phosphorylation following SST treatment. Inhibition of tyrosine phosphatase activity with sodium orthovanadate increased expression of the nicotinic acetyl-choline receptor (nAChR) epsilon subunit mRNA by three fold. Somatostatin reversed the elevated epsilon mRNA following orthovanadate treatment. These findings show that SSTR is expressed in skeletal muscle and that SST acting via the SSTR regulates tyrosine phosphorylation and expression of the epsilon subunit of the AChR in the rat skeletal muscle. PMID:9852305

  17. Characterization of urokinase receptor expression by human placental trophoblasts.

    PubMed

    Zini, J M; Murray, S C; Graham, C H; Lala, P K; Karikó, K; Barnathan, E S; Mazar, A; Henkin, J; Cines, D B; McCrae, K R

    1992-06-01

    The processes of implantation and placentation are both dependent on the invasion and remodeling of the uterine endometrium and vasculature by trophoblasts. Because the secretion and autocrine binding of urokinase (uPA) appears to be a common mechanism used by cells to facilitate plasmin-dependent tissue invasion, we measured the production of uPA and expression of uPA receptors by trophoblasts. Prourokinase bound specifically, reversibly, and with high affinity to cultured trophoblasts, via the uPA epidermal growth factor-like domain. Trophoblasts derived from two first-trimester placentae bound more prourokinase than cells isolated from term placentae. Furthermore, in vitro differentiation of cultured cytotrophoblasts into syncytiotrophoblasts was associated with diminished expression of urokinase receptors and a parallel decrease in the cellular content of uPA receptor mRNA. Trophoblasts also secreted prourokinase and plasminogen activator inhibitors types 1 and 2 (PAI-1 and PAI-2). Although prourokinase was secreted in amounts sufficient to endogenously saturate trophoblast uPA receptors, trophoblasts secreted greater amounts of PAI-1 and PAI-2 than uPA, and no net plasminogen activator activity was detected in trophoblast conditioned medium. In contrast, plasminogen added directly to cultured trophoblasts was readily converted to plasmin. Although the invasion and remodeling of uterine tissues by trophoblasts is a complex process dependent on several proteases of varying specificity, our findings suggest that the expression and modulation of urokinase receptors on the trophoblast cell surface may play an important role in this process. PMID:1316787

  18. Population-level control of gene expression

    NASA Astrophysics Data System (ADS)

    Nevozhay, Dmitry; Adams, Rhys; van Itallie, Elizabeth; Bennett, Matthew; Balazsi, Gabor

    2011-03-01

    Gene expression is the process that translates genetic information into proteins, that determine the way cells live, function and even die. It was demonstrated that cells with identical genomes exposed to the same environment can differ in their protein composition and therefore phenotypes. Protein levels can vary between cells due to the stochastic nature of intracellular biochemical events, indicating that the genotype-phenotype connection is not deterministic at the cellular level. We asked whether genomes could encode isogenic cell populations more reliably than single cells. To address this question, we built two gene circuits to control three cell population-level characteristics: gene expression mean, coefficient of variation and non-genetic memory of previous expression states. Indeed, we found that these population-level characteristics were more predictable than the gene expression of single cells in a well-controlled environment. This research was supported by the NIH Director's New Innovator Award 1DP2 OD006481-01 and Welch Foundation Grant C-1729.

  19. IONOTROPIC GLUTAMATE RECEPTORS mRNA EXPRESSION IN THE HUMAN THALAMUS: ABSENCE OF CHANGE IN SCHIZOPHRENIA

    PubMed Central

    Dracheva, Stella; Byne, William; Chin, Benjamin; Haroutunian, Vahram

    2009-01-01

    Abnormalities in glutamate neurotransmission are thought to be among the major contributing factors to the pathophysiology of schizophrenia. Although schizophrenia has been regarded mostly as a disorder of higher cortical function, the cortex and thalamus work as a functional unit. Existing data regarding alterations of glutamate receptor subunit expression in the thalamus in schizophrenia remain equivocal. This postmortem study examined mRNA expression of ionotropic glutamate receptor (iGluR) subunits and PSD95 in 5 precisely defined and dissected thalamic subdivisions (medial and lateral sectors of the mediodorsal nucleus; and the ventrolateral posterior, ventral posterior, and centromedian nuclei) of persons with schizophrenia and matched controls using quantitative PCR with normalization to multiple endogenous controls. Among 15 genes examined (NR1 and NR2A-D subunits of NMDA receptor; GluR1-4 subunits of AMPA receptor; GluR5-7 and KA1-2 subunits of kainate receptor; PSD95), all but two (GluR4 and KA1) were expressed at quantifiable levels. Differences in iGluR gene expression were seen between different nuclei but not between diagnostic groups. The relative abundance of transcripts was: NR1≫NR2A>NR2B>NR2D>NR2C for NMDA, GluR2>GluR1>GluR3 for AMPA, and KA2>GluR5>GluR7>GluR6 for kainate receptors. The expression of PSD95 correlated with the expression of NR1, NR2A, NR2B, NR2D and GluR6 in all nuclei. These results provide detailed and quantitative information on iGluR subunit expression in multiple nuclei of the human thalamus but suggest that alterations in their expression are not a prominent feature of schizophrenia. PMID:18462708

  20. Expression of prolactin receptors in normal canine mammary tissue, canine mammary adenomas and mammary adenocarcinomas

    PubMed Central

    2012-01-01

    Background Mammary tumors represent the most common neoplastic disease in female dogs. Recently, the promoting role of prolactin (PRL) in the development of human breast carcinoma has been shown. Possible proliferative, anti-apoptotic, migratory and angiogenic effects of PRL on human mammary cancer cells in vitro and in vivo were suggested. The effects of PRL are mediated by its receptor, and alterations in receptor expression are likely to play a role in tumor development. Currently, not much data is available about prolactin receptor (PRLR) expression in canine mammary tumors. To set the basis for investigations on the role of PRL in mammary tumorigenesis in this species, prolactin receptor expression was evaluated by semi-quantitative real time PCR and immunohistochemistry on 10 formalin-fixed, paraffin-embedded samples each of canine non-neoplastic mammary tissue, mammary adenomas and adenocarcinomas. Results The highest PRLR expression levels were found in normal mammary tissue, while adenomas, and to an even higher degree adenocarcinomas, showed a significant decrease in prolactin receptor expression. Compared to normal tissue, PRLR mRNA was reduced 2.4 fold (p = 0.0261) in adenomas and 4.8 fold (p = 0.008) in adenocarcinomas. PRLR mRNA expression was significantly lower in malignant than in benign lesions (p = 0.0165). Immunohistochemistry demonstrated PRLR expression in all three tissue types with signals mostly limited to epithelial cells. Conclusions Malignant transformation of mammary tissue was associated with a decline in prolactin receptor expression. Further studies are warranted to address the functional significance of this finding. PMID:22647582

  1. Expression of the Wnt Receptor Frizzled-4 in the Human Enteric Nervous System of Infants

    PubMed Central

    Nothelfer, Katharina; Obermayr, Florian; Belz, Nadine; Reinartz, Ellen; Bareiss, Petra M.; Bühring, Hans-Jörg; Beschorner, Rudi; Just, Lothar

    2016-01-01

    The Wnt signalling pathway plays a crucial role in the development of the nervous system. This signalling cascade is initiated upon binding of the secreted Wnt ligand to a member of the family of frizzled receptors. In the present study, we analysed the presence of frizzled-4 in the enteric nervous system of human infants. Frizzled-4 could be identified by immunohistochemistry in a subpopulation of enteric neuronal and glial cells in the small and large intestine. Detection of frizzled-4 in the tunica muscularis by RT-PCR confirmed this receptor's expression on the mRNA level. Interestingly, we observed distinct cell populations that co-expressed frizzled-4 with the intermediate filament protein nestin and the neurotrophin receptor p75NTR, which have been reported to be expressed in neural progenitor cells. Flow cytometry analysis revealed that 60% of p75NTR positive cells of the tunica muscularis were positive for frizzled-4. Additionally, in pathological samples of Hirschsprung's disease, the expression of this Wnt receptor correlated with the number of myenteric ganglion cells and decreased from normoganglionic to aganglionic areas of large intestine. The expression pattern of frizzled-4 indicates that this Wnt receptor could be involved in postnatal development and/or function of the enteric nervous system. PMID:26697080

  2. Altered expression of neuropeptide Y receptors caused by focal cortical dysplasia in human intractable epilepsy

    PubMed Central

    Luo, Hanjiang; Guan, Yuguang; Zhou, Jian; Qi, Xueling; Li, Tianfu; Xu, Zhiqing David; Luan, Guo-Ming

    2016-01-01

    Focal cortical dysplasia (FCD) is a common cause of pharmacologically-intractable epilepsy, however, the precise mechanisms underlying the epileptogenicity of FCD remains to be determined. Neuropeptide Y (NPY), an endogenous anticonvulsant in the central nervous system, plays an important role in the regulation of neuronal excitability. Increased expression of NPY and its receptors has been identified in the hippocampus of patients with mesial temporal lobe epilepsy, presumed to act as an endogenous anticonvulsant mechanism. Therefore, we investigated whether expression changes in NPY receptors occurs in patients with FCD. We specifically investigated the expression of seizure-related NPY receptor subtypes Y1, Y2, and Y5 in patients with FCD versus autopsy controls. We found that Y1R and Y2R were up-regulated at the mRNA and protein levels in the temporal and frontal lobes in FCD lesions. By contrast, there was no significant change in either receptor detected in parietal lesions. Notably, overexpression of Y5R was consistently observed in all FCD lesions. Our results demonstrate the altered expression of Y1R, Y2R and Y5R occurs in FCD lesions within the temporal, frontal and parietal lobe. Abnormal NPY receptor subtype expression may be associated with the onset and progression of epileptic activity and may act as a therapeutic candidate for the treatment of refractory epilepsy caused by FCD. PMID:26943580

  3. Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice

    PubMed Central

    Chen, Hsing-Jin; Chiang, Bor-Luen

    2015-01-01

    Background Preterm newborns that receive oxygen therapy often develop bronchopulmonary dysplasia (BPD), which is abnormal lung development characterized by impaired alveologenesis. Oxygen-mediated injury is thought to disrupt normal lung growth and development. However, the mechanism of hyperoxia-induced BPD has not been extensively investigated. We established a neonatal mouse model to investigate the effects of normobaric hyperoxia on retinoid metabolism and retinoid receptor expression. Methods Newborn mice were exposed to hyperoxic or normoxic conditions for 15 days. The concentration of retinol and retinyl palmitate in the lung was measured by HPLC to gauge retinoid metabolism. Retinoid receptor mRNA levels were assessed by real-time PCR. Proliferation and retinoid receptor expression in A549 cells were assessed in the presence and absence of exogenous vitamin A. Results Hyperoxia significantly reduced the body and lung weight of neonatal mice. Hyperoxia also downregulated expression of RARα, RARγ, and RXRγ in the lungs of neonatal mice. In vitro, hyperoxia inhibited proliferation and expression of retinoid receptors in A549 cells. Conclusion Hyperoxia disrupted retinoid receptor expression in neonatal mice. PMID:26509921

  4. Expression of androgen receptor splice variants in clinical breast cancers

    PubMed Central

    Dvinge, Heidi; Tarulli, Gerard A.; Hanson, Adrienne R.; Ryan, Natalie K.; Pickering, Marie A.; Birrell, Stephen N.; Hu, Dong Gui; Mackenzie, Peter I.; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V.; Dehm, Scott M.; Plymate, Stephen R.; Bradley, Robert K.; Tilley, Wayne D.; Selth, Luke A.

    2015-01-01

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer. PMID:26554309

  5. Expression of androgen receptor splice variants in clinical breast cancers.

    PubMed

    Hickey, Theresa E; Irvine, Connie M; Dvinge, Heidi; Tarulli, Gerard A; Hanson, Adrienne R; Ryan, Natalie K; Pickering, Marie A; Birrell, Stephen N; Hu, Dong Gui; Mackenzie, Peter I; Russell, Roslin; Caldas, Carlos; Raj, Ganesh V; Dehm, Scott M; Plymate, Stephen R; Bradley, Robert K; Tilley, Wayne D; Selth, Luke A

    2015-12-29

    The importance of androgen receptor (AR) signaling is increasingly being recognized in breast cancer, which has elicited clinical trials aimed at assessing the efficacy of androgen deprivation therapy (ADT) for metastatic disease. In prostate cancer, resistance to ADT is frequently associated with the emergence of androgen-independent splice variants of the AR (AR variants, AR-Vs) that lack the LBD and are constitutively active. Women with breast cancer may be prone to a similar phenomenon. Herein, we show that in addition to the prototypical transcript, the AR gene produces a diverse range of AR-V transcripts in primary breast tumors. The most frequently and highly expressed variant was AR-V7 (exons 1/2/3/CE3), which was detectable at the mRNA level in > 50% of all breast cancers and at the protein level in a subset of ERα-negative tumors. Functionally, AR-V7 is a constitutively active and ADT-resistant transcription factor that promotes growth and regulates a transcriptional program distinct from AR in ERα-negative breast cancer cells. Importantly, we provide ex vivo evidence that AR-V7 is upregulated by the AR antagonist enzalutamide in primary breast tumors. These findings have implications for treatment response in the ongoing clinical trials of ADT in breast cancer. PMID:26554309

  6. Role of Glucocorticoids in Cystic Ovarian Disease: Expression of Glucocorticoid Receptor in the Bovine Ovary.

    PubMed

    Amweg, Ayelen N; Rodríguez, Fernanda M; Huber, Emilia; Marelli, Belkis E; Salvetti, Natalia R; Rey, Florencia; Ortega, Hugo H

    2016-01-01

    The aim of this study was to characterize the expression of glucocorticoid receptor (GR) in the components of normal bovine ovary and in animals with cystic ovarian disease (COD). Changes in the protein and mRNA expression levels were determined in control cows and cows with COD by immunohistochemistry and real-time PCR. GR protein expression in granulosa cells was higher in cysts from animals with spontaneous COD and adrenocorticotropic hormone-induced COD than in tertiary follicles from control animals. In theca interna cells, GR expression was higher in cysts from animals with spontaneous COD than in tertiary follicles from control animals. The increase in GR expression observed in cystic follicles suggests a mechanism of action for cortisol and its receptor through the activation/inactivation of specific transcription factors. These factors could be related to the pathogenesis of COD in cattle. PMID:26677854

  7. Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses

    PubMed Central

    Xavier, Andre Machado; Anunciato, Aparecida Kataryna Olimpio; Rosenstock, Tatiana Rosado; Glezer, Isaias

    2016-01-01

    Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC’s effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive. PMID:27148162

  8. Gene Expression Control by Glucocorticoid Receptors during Innate Immune Responses.

    PubMed

    Xavier, Andre Machado; Anunciato, Aparecida Kataryna Olimpio; Rosenstock, Tatiana Rosado; Glezer, Isaias

    2016-01-01

    Glucocorticoids (GCs) are potent anti-inflammatory compounds that have been extensively used in clinical practice for several decades. GC's effects on inflammation are generally mediated through GC receptors (GRs). Signal transduction through these nuclear receptors leads to dramatic changes in gene expression programs in different cell types, typically due to GR binding to DNA or to transcription modulators. During the last decade, the view of GCs as exclusive anti-inflammatory molecules has been challenged. GR negative interference in pro-inflammatory gene expression was a landmark in terms of molecular mechanisms that suppress immune activity. In fact, GR can induce varied inhibitory molecules, including a negative regulator of Toll-like receptors pathway, or subject key transcription factors, such as NF-κB and AP-1, to a repressor mechanism. In contrast, the expression of some acute-phase proteins and other players of innate immunity generally requires GR signaling. Consequently, GRs must operate context-dependent inhibitory, permissive, or stimulatory effects on host defense signaling triggered by pathogens or tissue damage. This review aims to disclose how contradictory or comparable effects on inflammatory gene expression can depend on pharmacological approach (including selective GC receptor modulators; SEGRMs), cell culture, animal treatment, or transgenic strategies used as models. Although the current view of GR-signaling integrated many advances in the field, some answers to important questions remain elusive. PMID:27148162

  9. Problem-Solving Test: Expression Cloning of the Erythropoietin Receptor

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2008-01-01

    Terms to be familiar with before you start to solve the test: cytokines, cytokine receptors, cDNA library, cDNA synthesis, poly(A)[superscript +] RNA, primer, template, reverse transcriptase, restriction endonucleases, cohesive ends, expression vector, promoter, Shine-Dalgarno sequence, poly(A) signal, DNA helicase, DNA ligase, topoisomerases,…

  10. Antimullerian Hormone and Its Receptor Gene Expression in Prenatally Androgenized Female Rats

    PubMed Central

    Daneshian, Zahra; Ramezani Tehrani, Fahimeh; Zarkesh, Maryam; Norooz Zadeh, Mahsa; Mahdian, Reza; Zadeh Vakili, Azita

    2015-01-01

    Background: Anti-mullerian hormone (AMH) levels reflect the number of small antral follicles in ovaries and expression changes of AMH and its receptor are suspected to be involved in the pathogenesis of polycystic ovary syndrome (PCOS). Objectives: The aim of this study was to evaluate gene expression of AMH and its receptor in immature and adult rats prenatally exposed to androgen excess. Materials and Methods: Six pregnant Wistar rats in the experimental group were treated by subcutaneous injection of 5 mg free testosterone on day 20 of pregnancy, while controls (n = 6) received only 500 mL of solvent. Female pups of each mother were randomly divided into three groups as day 0 (newborn), 10-day old and days 75-85 (adult). RNAs were extracted from ovarian tissues and relative expression levels for AMH and its receptor genes were measured using TaqMan Real-Time PCR. Serum AMH and testosterone levels were measured using ELISA method. Results: Relative AMH expression decreased in newborns, 10-day olds and adults (0.806, 0.443 and 0.809 fold, respectively). AMHR expression was higher in newborns and adults (1.432 and 1.057 fold, respectively), while it decreased by 0.263 fold in 10-day olds, although none of them were significant (P > 0.05). In addition, AMH levels were consistent with the results of gene expression. Testosterone hormone levels from 10 day-olds to adults were significantly increased in both study groups (P = 0.016). Conclusions: While AMH receptor expression was higher in experimental rats, their serum concentrations of AMH were decreased. Further researches with greater sample sizes and measurement of bioactive forms of hormones are recommended to confirm the findings of this study. PMID:25745494

  11. Ovarian steroids regulate tachykinin and tachykinin receptor gene expression in the mouse uterus

    PubMed Central

    Pinto, Francisco M; Pintado, C Oscar; Pennefather, Jocelyn N; Patak, Eva; Candenas, Luz

    2009-01-01

    Background In the mouse uterus, pregnancy is accompanied by changes in tachykinin and tachykinin receptor gene expression and in the uterotonic effects of endogenous tachykinins. In this study we have investigated whether changes in tachykinin expression and responses are a result of changes in ovarian steroid levels. Methods We quantified the mRNAs of tachykinins and tachykinin receptors in uteri from ovariectomized mice and studied their regulation in response to estrogen and progesterone using real-time quantitative RT-PCR. Early (3 h) and late (24 h) responses to estrogen were evaluated and the participation of the estrogen receptors (ER), ERalpha and ERbeta, was analyzed by treating mice with propylpyrazole triol, a selective ERalpha agonist, or diarylpropionitrile, a selective agonist of ERbeta. Results All genes encoding tachykinins (Tac1, Tac2 and Tac4) and tachykinin receptors (Tacr1, Tacr2 and Tacr3) were expressed in uteri from ovariectomized mice. Estrogen increased Tac1 and Tacr1 mRNA after 3 h and decreased Tac1 and Tac4 expression after 24 h. Tac2 and Tacr3 mRNA levels were decreased by estrogen at both 3 and 24 h. Most effects of estrogen were also observed in animals treated with propylpyrazole triol. Progesterone treatment increased the levels of Tac2. Conclusion These results show that the expression of tachykinins and their receptors in the mouse uterus is tightly and differentially regulated by ovarian steroids. Estrogen effects are mainly mediated by ERalpha supporting an essential role for this estrogen receptor in the regulation of the tachykinergic system in the mouse uterus. PMID:19627578

  12. Characterization of p75 neurotrophin receptor expression in human dental pulp stem cells.

    PubMed

    Pan, Wenru; Kremer, Karlea L; Kaidonis, Xenia; Ludlow, Victoria E; Rogers, Mary-Louise; Xie, Jianling; Proud, Christopher G; Koblar, Simon A

    2016-10-01

    Human adult dental pulp stem cells (DPSC) are a heterogeneous stem cell population, which are able to differentiate down neural, chondrocyte, osteocyte and adipocyte lineages. We studied the expression pattern of p75 neurotrophin receptors (p75NTR), a marker of neural stem cells, within human DPSC populations from eight donors. p75NTR are expressed at low levels (<10%) in DPSC. Importantly, p75(+) DPSC represent higher expression levels of SOX1 (neural precursor cell marker), SOX2 (cell pluripotency marker) and nestin (neural stem cell marker) in comparison to p75(-) DPSC. Our results suggest that p75(+) hDPSC may denote a subpopulation with greater neurogenic potential. PMID:27469433

  13. Evaluation of leptin receptor expression on buffalo leukocytes.

    PubMed

    De Matteis, Giovanna; Grandoni, Francesco; Scatà, Maria Carmela; Catizone, Angela; Reale, Anna; Crisà, Alessandra; Moioli, Bianca

    2016-09-01

    Experimental evidences support a direct role for leptin in immunity. Besides controlling food intake and energy expenditure, leptin was reported to be involved in the regulation of the immune system in ruminants. The aim of this work was to highlight the expression of leptin receptor (LEPR) on Bubalus bubalis immune cells using a multi-approach assessment: flow cytometry, confocal microscopy and gene expression analysis. Flow cytometric analysis of LEPR expression showed that peripheral blood monocytes were the predominant cells expressing LEPR. This result was corroborated by confocal microscopy and RT-PCR analysis. Moreover, among lymphocytes, LEPR was mainly expressed by B lymphocytes and Natural Killer cells. Evidence of LEPR expression on buffalo blood leukocytes showed to be a good indicator of the responsivity of these cells to leptin, so confirming the involvement of leptin in buffalo immune response. PMID:27436440

  14. Structure and regulation of expression of the mouse GH receptor.

    PubMed

    Talamantes, F; Ortiz, R

    2002-10-01

    GH-binding protein (GHBP) in the mouse consists of a ligand-binding domain, which is identical to the extracellular portion of the GH receptor (GHR), and a hydrophilic C-terminal domain, in place of the transmembrane and intracellular domains of the GHR. The two proteins are encoded by separate mRNAs which are derived from a single gene by alternative splicing. Determination of the gestational profiles of GHR and GHBP mRNA expression in mouse liver and placenta shows that in the liver, the 1.4 kb mRNA corresponding to the mouse GHBP increases approximately 20-fold between non-pregnant and late pregnant mice, whereas the relative increase in the expression of the 4.2 kb mouse GHR was 8-fold. The rise in the steady-state levels of both mRNAs began on day 9 of gestation. Mouse GHBP mRNA levels continue to rise until day 15 of pregnancy, while GHR mRNA abundance reaches a plateau by day 13. By elucidating the temporal changes in GHR and GHBP mRNA abundance during pregnancy and lactation in multiple maternal tissues and by assessing the ontogeny of these mRNAs in fetal and early postnatal mouse liver, our studies have demonstrated that the alternative splicing of mouse GHR/GHBP mRNA precursor is regulated in a tissue-, developmental stage- and physiological state-specific manner. In vitro studies using hepatocytes in culture have begun to elucidate the hormonal factor(s) involved in the gestation control of the expression of GHR and GHBP. Treatment of hepatocytes with GH or estradiol (E2) alone did not have any effect on the cellular concentrations of GHBP and GHR. However, the combination of E2 and GH up-regulated the cellular concentrations of GHBP and GHR 2- to 3-fold. GHBP and GHR mRNA concentrations were also up-regulated 2- to 3-fold. ICI 182-780, a competitive inhibitor of E2 for the estrogen receptor (ER), at different concentrations inhibited the E2- and GH-induced stimulation of GHBP and GHR. Furthermore, ER concentrations increased 5- to 7-fold in hepatocytes

  15. Estrogen Receptor (ER) β Regulates ERα Expression in Stromal Cells Derived from Ovarian Endometriosis

    PubMed Central

    Trukhacheva, Elena; Lin, Zhihong; Reierstad, Scott; Cheng, You-Hong; Milad, Magdy; Bulun, Serdar E.

    2009-01-01

    Context: Estradiol and its nuclear receptors, estrogen receptor (ER) α and ERβ, play critical roles in endometrium and endometriosis. Levels of ERβ, due to pathological hypomethylation of its promoter, are significantly higher in endometriotic vs. endometrial tissue and stromal cells, whereas ERα levels are lower in endometriosis. Estradiol regulates ERα gene expression via its alternatively used promoters A, B, and C. Objective: The aim of the study was to determine whether high levels of ERβ in endometriotic stromal cells from ovarian endometriomas regulate ERα gene expression. Results: ERβ knockdown significantly increased ERα mRNA and protein levels in endometriotic stromal cells. Conversely, ERβ overexpression in endometrial stromal cells decreased ERα mRNA and protein levels. ERβ knockdown significantly decreased proliferation of endometriotic stromal cells. Chromatin immunoprecipitation assays demonstrated that estradiol enhanced ERβ binding to nonclassical activator protein 1 and specificity protein 1 motifs in the ERα gene promoters A and C and a classic estrogen response element in promoter B in endometriotic stromal cells. Conclusions: High levels of ERβ suppress ERα expression and response to estradiol in endometrial and endometriotic stromal cells via binding to classic and nonclassic DNA motifs in alternatively used ERα promoters. ERβ also regulates cell cycle progression and might contribute to proliferation of endometriotic stromal cells. We speculate that a significantly increased ratio of ERβ:ERα in endometriotic tissues may also suppress progesterone receptor expression and contribute to progesterone resistance. Thus, ERβ may serve as a significant therapeutic target for endometriosis. PMID:19001520

  16. Expression and function of a novel variant of estrogen receptor-α36 in murine airways.

    PubMed

    Jia, Shuping; Zhang, Xintian; He, David Z Z; Segal, Manav; Berro, Abdo; Gerson, Trevor; Wang, Zhaoyi; Casale, Thomas B

    2011-11-01

    Evidence suggests that estrogen signaling is involved in sex differences in the prevalence rates and control of asthma, but the expression patterns of estrogen receptor variants and estrogen function in the lung are not well established. We investigated the expression of major estrogen receptor variants occurring naturally and after the development of allergen-induced airway hyperreactivity in a murine model of allergic asthma, along with the role of estrogen signaling in small-airway ciliary motion and smooth muscle contraction. Female BALB/c mice were sensitized with ovalbumin, and estrogen receptor expression patterns were examined by immunofluorescence and Western blot analysis. Time-lapse video and photodiode-based displacement measurement systems were used to assess the effects of estrogen signaling on airway ciliary beat frequency and smooth muscle contraction. We found that a novel variant of estrogen receptor (ER)-α, ER-α36, is expressed in airway epithelial and smooth muscle cells. ER-α36 was predominately localized on the plasma membranes of airway cells. After sensitization to allergen, the expression levels of ER-α36 increased significantly (P < 0.01), whereas the expression of ER-β and ER-α66 did not significantly change. Estrogen treatment in vitro resulted in a rapid increase in airway cilia motion in a dose-dependent fashion, but did not exert any effect on airway smooth muscle contraction. We speculate that the up-regulation of estrogen receptor expression associated with allergen-induced airway hyperresponsiveness may constitute a protective mechanism to facilitate the clearance of mucus. The identification and localization of specific estrogen receptor subtypes in the lung could lead to newer therapeutic avenues aimed at addressing sex differences of asthma susceptibility. PMID:21642591

  17. Expression of adenosine A2b receptor in rat type II and III taste cells.

    PubMed

    Nishida, Kentaro; Dohi, Yukari; Yamanaka, Yuri; Miyata, Ai; Tsukamoto, Katsunobu; Yabu, Miharu; Ohishi, Akihiro; Nagasawa, Kazuki

    2014-05-01

    We previously demonstrated that equilibrative nucleoside transporter 1 was expressed in taste cells, suggesting the existence of an adenosine signaling system, but whether or not the expression of an adenosine receptor occurs in rat taste buds remains unknown. Therefore, we examined the expression profiles of adenosine receptors and evaluated their functionality in rat circumvallate papillae. Among adenosine receptors, the mRNA for an adenosine A2b receptor (A2bR) was expressed by the rat circumvallate papillae, and its expression level was significantly greater in the circumvallate papillae than in the non-taste lingual epithelium. A2bR-immunoreactivity was detected primarily in type II taste cells, and partial, but significant expression was also observed in type III ones, but there was no immunoreactivity in type I ones. The cAMP generation in isolated epithelium containing taste buds treated with 500 μM adenosine or 10 μM BAY60-6583 was significantly increased compared to in the controls. These findings suggest that adenosine plays a role in signaling transmission via A2bR between taste cells in rats. PMID:24327108

  18. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis.

    PubMed

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  19. Prognostic Relevance of Cytokine Receptor Expression in Acute Myeloid Leukemia: Interleukin-2 Receptor α-Chain (CD25) Expression Predicts a Poor Prognosis

    PubMed Central

    Nakase, Kazunori; Kita, Kenkichi; Kyo, Taiichi; Ueda, Takanori; Tanaka, Isao; Katayama, Naoyuki

    2015-01-01

    A variety of cytokine/cytokine receptor systems affect the biological behavior of acute leukemia cells. However, little is known about the clinical relevance of cytokine receptor expression in acute myeloid leukemia (AML). We quantitatively examined the expression of interleukin-2 receptor α-chain (IL-2Rα, also known as CD25), IL-2Rβ, IL-3Rα, IL-4Rα, IL-5Rα, IL-6Rα, IL-7Rα, the common β-chain (βc), γc, granulocyte-macrophage colony-stimulating factor (GM-CSF)Rα, G-CSFR, c-fms, c-mpl, c-kit, FLT3, and GP130 in leukemia cells from 767 adult patients with AML by flow cytometry and determined their prevalence and clinical significance. All cytokine receptors examined were expressed at varying levels, whereas the levels of IL-3Rα, GM-CSFRα, IL-2Rα, γc, c-kit, and G-CSFR exhibited a wide spectrum of ≥10,000 sites/cell. In terms of their French-American-British classification types, GM-CSFRα and c-fms were preferentially expressed in M4/M5 patients, G-CSF in M3 patients, and IL-2Rα in non-M3 patients. Elevated levels of IL-3Rα, GM-CSFRα, and IL-2Rα correlated with leukocytosis. In patients ≤60 years old, higher levels of these 3 receptors correlated with poor responses to conventional chemotherapy, but only IL-2Rα was associated with a shorter overall survival. By incorporating IL-2Rα status into cytogenetic risk stratification, we could sort out a significantly adverse-risk cohort from the cytogenetically intermediate-risk group. Analyses with various phenotypical risk markers revealed the expression of IL-2Rα as an independent prognostic indicator in patients with intermediate-risk cytogenetics. These findings were not observed in patients >60 years old. Our results indicate that several cytokine receptors were associated with certain cellular and clinical features, but IL-2Rα alone had prognostic value that provides an additional marker to improve current risk evaluation in AML patients ≤60 years old. PMID:26375984

  20. Molecular cloning and functional expression of a brain-specific somatostatin receptor.

    PubMed Central

    Bruno, J F; Xu, Y; Song, J; Berelowitz, M

    1992-01-01

    The PCR and conventional library screening were used to clone the brain-specific somatostatin receptor rSSTR-4 from a rat genomic library. The deduced amino acid sequence encodes a protein of 384 amino acids and displays structural and sequence homologies with members of the G protein-receptor superfamily. The amino acid sequence of rSSTR-4 is 60% and 48% identical to that of somatostatin receptors SSTR-1 and SSTR-2, respectively, two recently cloned subtypes. Competition curve analysis of the binding properties of the receptor transiently expressed in COS-1 cells revealed a higher apparent affinity for somatostatin 14 than for somatostatin 28. In contrast, the somatostatin analogs SMS 201-995, IM 4-28, and MK-678 failed to displace specific binding in transfected cells. These characteristics resemble the pharmacological binding properties of the previously described brain-specific somatostatin-receptor subtype. Examination of the tissue distribution of mRNA for rSSTR-4 revealed expression limited to various brain regions with highest levels in the cortex and hippocampus. Thus, based on the pharmacology and tissue localization of this receptor, we conclude that rSSTR-4 represents a brain-specific somatostatin receptor. Images PMID:1360663

  1. Expression of serotonin receptor genes in cranial ganglia.

    PubMed

    Maeda, Naohiro; Ohmoto, Makoto; Yamamoto, Kurumi; Kurokawa, Azusa; Narukawa, Masataka; Ishimaru, Yoshiro; Misaka, Takumi; Matsumoto, Ichiro; Abe, Keiko

    2016-03-23

    Taste cells release neurotransmitters to gustatory neurons to transmit chemical information they received. Sweet, umami, and bitter taste cells use ATP as a neurotransmitter. However, ATP release from sour taste cells has not been observed so far. Instead, they release serotonin when they are activated by sour/acid stimuli. Thus it is still controversial whether sour taste cells use ATP, serotonin, or both. By reverse transcription-polymerase chain reaction and subsequent in situ hybridization (ISH) analyses, we revealed that of 14 serotonin receptor genes only 5-HT3A and 5-HT3B showed significant/clear signals in a subset of neurons of cranial sensory ganglia in which gustatory neurons reside. Double-fluorescent labeling analyses of ISH for serotonin receptor genes with wheat germ agglutinin (WGA) in cranial sensory ganglia of pkd1l3-WGA mice whose sour neural pathway is visualized by the distribution of WGA originating from sour taste cells in the posterior region of the tongue revealed that WGA-positive cranial sensory neurons rarely express either of serotonin receptor gene. These results suggest that serotonin receptors expressed in cranial sensory neurons do not play any role as neurotransmitter receptor from sour taste cells. PMID:26854841

  2. Expression of neuropeptide hormone receptors in human adrenal tumors and cell lines: antiproliferative effects of peptide analogues.

    PubMed

    Ziegler, C G; Brown, J W; Schally, A V; Erler, A; Gebauer, L; Treszl, A; Young, L; Fishman, L M; Engel, J B; Willenberg, H S; Petersenn, S; Eisenhofer, G; Ehrhart-Bornstein, M; Bornstein, S R

    2009-09-15

    Peptide analogues targeting various neuropeptide receptors have been used effectively in cancer therapy. A hallmark of adrenocortical tumor formation is the aberrant expression of peptide receptors relating to uncontrolled cell proliferation and hormone overproduction. Our microarray results have also demonstrated a differential expression of neuropeptide hormone receptors in tumor subtypes of human pheochromocytoma. In light of these findings, we performed a comprehensive analysis of relevant receptors in both human adrenomedullary and adrenocortical tumors and tested the antiproliferative effects of peptide analogues targeting these receptors. Specifically, we examined the receptor expression of somatostatin-type-2 receptor, growth hormone-releasing hormone (GHRH) receptor or GHRH receptor splice variant-1 (SV-1) and luteinizing hormone-releasing hormone (LHRH) receptor at the mRNA and protein levels in normal human adrenal tissues, adrenocortical and adrenomedullary tumors, and cell lines. Cytotoxic derivatives of somatostatin AN-238 and, to a lesser extent, AN-162, reduced cell numbers of uninduced and NGF-induced adrenomedullary pheochromocytoma cells and adrenocortical cancer cells. Both the splice variant of GHRH receptor SV-1 and the LHRH receptor were also expressed in adrenocortical cancer cell lines but not in the pheochromocytoma cell line. The GHRH receptor antagonist MZ-4-71 and LHRH antagonist Cetrorelix both significantly reduced cell growth in the adrenocortical cancer cell line. In conclusion, the expression of receptors for somatostatin, GHRH, and LHRH in the normal human adrenal and in adrenal tumors, combined with the growth-inhibitory effects of the antitumor peptide analogues, may make possible improved treatment approaches to adrenal tumors. PMID:19717419

  3. Expression of c-kit receptor and its autophosphorylation in immature rat type A spermatogonia.

    PubMed

    Dym, M; Jia, M C; Dirami, G; Price, J M; Rabin, S J; Mocchetti, I; Ravindranath, N

    1995-01-01

    The objective of this study was to examine the expression and activation of the c-kit receptor, a specific receptor for kit ligand (stem cell factor, steel factor), in rat type A spermatogonia. Testes were obtained from 9-day-old rats, decapsulated, and then subjected to sequential enzymatic digestion. The mixture of testicular cell types was then separated by sedimentation velocity at unit gravity. The isolated type A spermatogonia were characterized by light and electron microscopy. They exhibited spherical nuclei containing several nucleoli and associated chromatin clumps and organelles generally in a perinuclear location similar to that found in the in vivo 9-day-old testis. The synthesis of the c-kit receptor by the spermatogonia was established by hybridization of total RNA with a specific cDNA for mouse c-kit receptor. Two mRNA transcripts migrating at 4.8 kb and 12 kb were observed. Localization of the c-kit receptor in the isolated cells was determined by immunocytochemistry using an antibody to c-kit protein. Specific staining for c-kit receptor was observed in the cytoplasm of the isolated type A spermatogonia. Furthermore, the presence of the c-kit receptor protein in the spermatogonia was confirmed by Western blot analysis using the same antibody. The antibody recognized the c-kit receptor at approximately 160 kDa. In an attempt to determine whether this receptor has a functional significance, we examined the effect of kit ligand on the phosphorylation of the c-kit receptor. The c-kit receptor appeared to be constitutively autophosphorylated on tyrosine at low basal levels, and upon stimulation with kit ligand, the amount of phosphorylated protein increased significantly. These observations indicate that kit ligand induces autophosphorylation of the c-kit receptor, which may lead to the activation of other cellular target proteins responsible for spermatogonial proliferation and/or differentiation. PMID:7536046

  4. Elevated Dengue Virus Nonstructural Protein 1 Serum Levels and Altered Toll-Like Receptor 4 Expression, Nitric Oxide, and Tumor Necrosis Factor Alpha Production in Dengue Hemorrhagic Fever Patients

    PubMed Central

    Carvalho, Denise Maciel; Garcia, Fernanda Gonçalves; Terra, Ana Paula Sarreta; Lopes Tosta, Ana Cristina; Silva, Luciana de Almeida; Castellano, Lúcio Roberto; Silva Teixeira, David Nascimento

    2014-01-01

    Background. During dengue virus (DV) infection, monocytes produce tumor necrosis factor alpha (TNF-α) and nitric oxide (NO) which might be critical to immunopathogenesis. Since intensity of DV replication may determine clinical outcomes, it is important to know the effects of viral nonstructural protein 1 (NS1) on innate immune parameters of infected patients. The present study investigates the relationships between dengue virus nonstructural protein 1 (NS1) serum levels and innate immune response (TLR4 expression and TNF-α/NO production) of DV infected patients presenting different clinical outcomes. Methodology/Principal Findings. We evaluated NO, NS1 serum levels (ELISA), TNF-α production by peripheral blood mononuclear cells (PBMCs), and TLR4 expression on CD14+ cells from 37 dengue patients and 20 healthy controls. Early in infection, increased expression of TLR4 in monocytes of patients with dengue fever (DF) was detected compared to patients with dengue hemorrhagic fever (DHF). Moreover, PBMCs of DHF patients showed higher NS1 and lower NO serum levels during the acute febrile phase and a reduced response to TLR4 stimulation by LPS (with a reduced TNF-α production) when compared to DF patients. Conclusions/Significance. During DV infection in humans, some innate immune parameters change, depending on the NS1 serum levels, and phase and severity of the disease which may contribute to development of different clinical outcomes. PMID:25580138

  5. Identification and Differential Expression of a Candidate Sex Pheromone Receptor in Natural Populations of Spodoptera litura

    PubMed Central

    Lin, Xinda; Zhang, Qinhui; Wu, Zhongnan; Du, Yongjun

    2015-01-01

    Olfaction is primarily mediated by highly specific olfactory receptors (ORs), a subfamily of which are the pheromone receptors that play a key role in sexual communication and can contribute to reproductive isolation. Here we cloned and identified an olfactory receptor, SlituOR3 (Genbank NO. JN835270), from Spodoptera litura, to be the candidate pheromone receptor. It exhibited male-biased expression in the antennae, where they were localized at the base of sensilla trichoidea. Conserved orthologues of these receptors were found amongst known pheromone receptors within the Lepidoptera, and SlituOR3 were placed amongst a clade of candidate pheromone receptors in a phylogeny tree of insect ORs. SlituOR3 is required for the EAG responses to both Z9E11-14:OAc and Z9E12-14:OAc SlituOR3 showed differential expression in S. litura populations attracted to traps baited with a series of sex pheromone blends composed of different ratios of (9Z,11E)-tetradecadienyl acetate (Z9E11-14:OAc) and (9Z,12E)-tetradecadienyl acetate (Z9E12-14:OAc). The changes in the expression level of SlitOR3 and antennal responses after SlitOR3 silencing suggested that SlitOR3 is required for the sex pheromone signaling. We infer that variation in transcription levels of olfactory receptors may modulate sex pheromone perception in male moths and could affect both of pest control and monitoring efficiency by pheromone application after long time mass trapping with one particular ratio of blend in the field. PMID:26126192

  6. Expression and functional study of estrogen receptor-related receptors in human prostatic cells and tissues.

    PubMed

    Cheung, C P; Yu, Shan; Wong, K B; Chan, L W; Lai, Fernand M M; Wang, Xianghong; Suetsugi, Masatomo; Chen, Shiuan; Chan, Franky L

    2005-03-01

    Estrogen receptor-related receptors (ERRs; alpha, beta, gamma) are orphan nuclear receptors and constitutively active without binding to estrogen. Like estrogen receptors (ERs), ERRs bind to estrogen receptor elements and estrogen receptor element-related repeats. Growing evidence suggests that ERRs can cross-talk with ERs in different cell types via competition for DNA sites and coactivators. We hypothesize that ERRs might play regulatory roles in normal and neoplastic prostatic cells by sharing similar ER-mediated pathways or acting independently. In this study, we investigated mRNA and protein expression patterns of three ERR members in normal human prostate epithelial cells, established cell lines, cancer xenografts, and prostatic tissues. Additionally, effects of transient transfection of ERRs on prostatic cell proliferation and ER expression were also examined. RT-PCR showed that ERRalpha and ERRgamma transcripts were detected in most cell lines and xenografts, whereas ERRbeta was detected in normal epithelial cells and few immortalized cell lines but not in most cancer lines. Similar results were demonstrated in clinical prostatic specimens. Western blottings and immunohistochemistry confirmed similar expression patterns that ERR proteins were detected as nuclear proteins in epithelial cells, whereas their expressions became reduced or undetected in neoplastic prostatic cells. Transient transfection confirmed that ERRs were expressed in prostatic cells as nuclear proteins and transcriptionally active in the absence of estradiol. Transfection results showed that overexpression of ERRs inhibited cell proliferation and repressed ERalpha transcription in PC-3 cells. Our study shows that ERRs, which are coexpressed with ERs in prostatic cells, could regulate cell growth and modulate ER-mediated pathways via interference on ERalpha transcription in prostatic cells. PMID:15598686

  7. Aromatase and estrogen receptor alpha mRNA expression as prognostic biomarkers in patients with astrocytomas.

    PubMed

    Dueñas Jiménez, J M; Candanedo Arellano, A; Santerre, A; Orozco Suárez, S; Sandoval Sánchez, H; Feria Romero, I; López-Elizalde, R; Alonso Venegas, M; Netel, B; de la Torre Valdovinos, B; Dueñas Jiménez, S H

    2014-09-01

    Estrogens are oncogenic hormones at a high level in breast, prostate, endometrial and lung cancer. Estrogens are synthesized by aromatase which has been used as a biomarker both in breast and lung cancer. Estrogen biological activities are executed by their classic receptors (ERα and ERβ). ERα has been described as a cancer promoter and ERβ, as a possible tumor suppressor. Both receptors are present at low levels in primary multiforme glioblastoma (GBM). The GBM frequency is 50 % higher in men than in women. The GBM patient survival period ranges from 7 to 18 months. The purpose of this pilot study was to evaluate aromatase and estrogen receptor expression, as well as 17ß-estradiol concentration in astrocytoma patients biopsies to obtain a prognosis biomarker for these patients. We analyzed 36 biopsies of astrocytoma patients with a different grade (I-IV) of malignity. Aromatase and estrogen receptor mRNA expression were analyzed by semiquantitative RT-PCR, and the E2 levels, by ELISA. E2 concentration was higher in GBM, compared to grade II or III astrocytomas. The number of cells immunoreactive to aromatase and estrogen receptors decreased as the grade of tumor malignity increased. Aromatase mRNA expression was present in all biopsies, regardless of malignity grade or patient age or gender. The highest expression of aromatase mRNA in GBM patients was associated to the worst survival prognostic (6.28 months). In contrast lowest expression of ERα mRNA in astrocytoma patients had a worst prognosis. In conclusion, aromatase and ERα expression could be used as prognosis biomarkers for astrocytoma patients. PMID:25005528

  8. Developmental regulation of N-methyl-D-aspartate- and kainate-type glutamate receptor expression in the rat spinal cord

    NASA Technical Reports Server (NTRS)

    Stegenga, S. L.; Kalb, R. G.

    2001-01-01

    Spinal motor neurons undergo experience-dependent development during a critical period in early postnatal life. It has been suggested that the repertoire of glutamate receptor subunits differs between young and mature motor neurons and contributes to this activity-dependent development. In the present study we examined the expression patterns of N-methyl-D-aspartate- and kainate-type glutamate receptor subunits during the postnatal maturation of the spinal cord. Young motor neurons express much higher levels of the N-methyl-D-aspartate receptor subunit NR1 than do adult motor neurons. Although there are eight potential splice variants of NR1, only a subgroup is expressed by motor neurons. With respect to NR2 receptor subunits, young motor neurons express NR2A and C, while adult motor neurons express only NR2A. Young motor neurons express kainate receptor subunits GluR5, 6 and KA2 but we are unable to detect these or any other kainate receptor subunits in the adult spinal cord. Other spinal cord regions display a distinct pattern of developmental regulation of N-methyl-D-aspartate and kainate receptor subunit expression in comparison to motor neurons. Our findings indicate a precise spatio-temporal regulation of individual subunit expression in the developing spinal cord. Specific combinations of subunits in developing neurons influence their excitable properties and could participate in the emergence of adult neuronal form and function.

  9. Expression of leptin and leptin receptor isoforms in the rat and human carotid body.

    PubMed

    Porzionato, Andrea; Rucinski, Marcin; Macchi, Veronica; Stecco, Carla; Castagliuolo, Ignazio; Malendowicz, Ludwik K; De Caro, Raffaele

    2011-04-18

    Leptin is known to play a role in the modulation of metabolism and control of breathing acting mainly on central nervous structures, although additional actions on peripheral arterial chemoreceptors have also been suggested in the literature. We therefore examined by means of immunohistochemistry the expression of leptin and leptin receptors in the carotid bodies of rats and humans. Leptin expression and relative expression of leptin receptor isoforms were also studied in rats by real-time PCR. No leptin or leptin receptor immunoreactivities were visible in the type II cells of either series. In rat carotid bodies, diffuse positive stainings for leptin and leptin receptors (both with antibody recognizing all receptor isoforms and antibody specific for Ob-Rb) were observed in type I cells. In human carotid bodies, the mean percentage (±standard error) of leptin immunoreactive type I cells was 39.4%±5.1% and the percentages of leptin receptor immunoreactive type I cells were 57.3%±3.9% with antibody recognizing all receptor isoforms and 33.3%±4.2% with antibody specific for isoform Ob-Rb. Double immunofluorescences with anti-tyrosine hydroxylase (type I cell marker) and anti-glial fibrillary acidic protein (type II cell markers) confirmed the selective location of leptin and Ob-Rb in type I cells. Real-time PCR showed the expression of leptin and Ob-Ra, Ob-Rb, Ob-Rc and Ob-Rf isoform mRNA in the rat carotid body, levels of expression being Ob-Rf>Ob-Rc>Ob-Ra>Ob-Rb. Ob-Re mRNA was not detected. The above findings suggest a role of circulating or locally produced leptin in the regulation of chemoreceptor discharge and/or metabolic sensing function, by means of direct action on type I cells. PMID:21334312

  10. Transient Receptor Potential Canonical 1 (TRPC1) Channels as Regulators of Sphingolipid and VEGF Receptor Expression

    PubMed Central

    Asghar, Muhammad Yasir; Magnusson, Melissa; Kemppainen, Kati; Sukumaran, Pramod; Löf, Christoffer; Pulli, Ilari; Kalhori, Veronica; Törnquist, Kid

    2015-01-01

    The identity of calcium channels in the thyroid is unclear. In human follicular thyroid ML-1 cancer cells, sphingolipid sphingosine 1-phosphate (S1P), through S1P receptors 1 and 3 (S1P1/S1P3), and VEGF receptor 2 (VEGFR2) stimulates migration. We show that human thyroid cells express several forms of transient receptor potential canonical (TRPC) channels, including TRPC1. In TRPC1 knockdown (TRPC1-KD) ML-1 cells, the basal and S1P-evoked invasion and migration was attenuated. Furthermore, the expression of S1P3 and VEGFR2 was significantly down-regulated. Transfecting wild-type ML-1 cells with a nonconducting TRPC1 mutant decreased S1P3 and VEGFR2 expression. In TRPC1-KD cells, receptor-operated calcium entry was decreased. To investigate whether the decreased receptor expression was due to attenuated calcium entry, cells were incubated with the calcium chelator BAPTA-AM (1,2-bis(o-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid). In these cells, and in cells where calmodulin and calmodulin-dependent kinase were blocked pharmacologically, S1P3 and VEGFR2 expression was decreased. In TRPC1-KD cells, both hypoxia-inducible factor 1α expression and the secretion and activity of MMP2 and MMP9 were attenuated, and proliferation was decreased in TRPC1-KD cells. This was due to a prolonged G1 phase of the cell cycle, a significant increase in the expression of the cyclin-dependent kinase inhibitors p21 and p27, and a decrease in the expression of cyclin D2, cyclin D3, and CDK6. Transfecting TRPC1 to TRPC1-KD cells rescued receptor expression, migration, and proliferation. Thus, the expression of S1P3 and VEGFR2 is mediated by a calcium-dependent mechanism. TRPC1 has a crucial role in this process. This regulation is important for the invasion, migration, and proliferation of thyroid cancer cells. PMID:25971967

  11. Effects of retinoic acid on growth hormone-releasing hormone receptor, growth hormone secretagogue receptor gene expression and growth hormone secretion in rat anterior pituitary cells.

    PubMed

    Maliza, Rita; Fujiwara, Ken; Tsukada, Takehiro; Azuma, Morio; Kikuchi, Motoshi; Yashiro, Takashi

    2016-06-30

    Retinoic acid (RA) is an important signaling molecule in embryonic development and adult tissue. The actions of RA are mediated by the nuclear receptors retinoic acid receptor (RAR) and retinoid X receptor (RXR), which regulate gene expression. RAR and RXR are widely expressed in the anterior pituitary gland. RA was reported to stimulate growth hormone (GH) gene expression in the anterior pituitary cells. However, current evidence is unclear on the role of RA in gene expression of growth hormone-releasing hormone receptor (Ghrh-r), growth hormone secretagogue receptor (Ghs-r) and somatostatin receptors (Sst-rs). Using isolated anterior pituitary cells of rats, we examined the effects of RA on gene expression of these receptors and GH release. Quantitative real-time PCR revealed that treatment with all-trans retinoic acid (ATRA; 10(-6) M) for 24 h increased gene expression levels of Ghrh-r and Ghs-r; however, expressions of Sst-r2 and Sst-r5 were unchanged. Combination treatment with the RAR-agonist Am80 and RXR-agonist PA024 mimicked the effects of ATRA on Ghrh-r and Ghs-r gene expressions. Exposure of isolated pituitary cells to ATRA had no effect on basal GH release. In contrast, ATRA increased growth hormone-releasing hormone (GHRH)- and ghrelin-stimulated GH release from cultured anterior pituitary cells. Our results suggest that expressions of Ghrh-r and Ghs-r are regulated by RA through the RAR-RXR receptor complex and that RA enhances the effects of GHRH and ghrelin on GH release from the anterior pituitary gland. PMID:27052215

  12. Surface expression of NMDA receptor changes during memory consolidation in the crab Neohelice granulata.

    PubMed

    Hepp, Yanil; Salles, Angeles; Carbo-Tano, Martin; Pedreira, Maria Eugenia; Freudenthal, Ramiro

    2016-08-01

    The aim of the present study was to analyze the surface expression of the NMDA-like receptors during the consolidation of contextual learning in the crab Neohelice granulata Memory storage is based on alterations in the strength of synaptic connections between neurons. The glutamatergic synapses undergo various forms of N-methyl-D aspartate receptor (NMDAR)-dependent changes in strength, a process that affects the abundance of other receptors at the synapse and underlies some forms of learning and memory. Here we propose a direct regulation of the NMDAR. Changes in NMDAR's functionality might be induced by the modification of the subunit's expression or cellular trafficking. This trafficking does not only include NMDAR's movement between synaptic and extra-synaptic localizations but also the cycling between intracellular compartments and the plasma membrane, a process called surface expression. Consolidation of contextual learning affects the surface expression of the receptor without affecting its general expression. The surface expression of the GluN1 subunit of the NMDAR is down-regulated immediately after training, up-regulated 3 h after training and returns to naïve and control levels 24 h after training. The changes in NMDAR surface expression observed in the central brain are not seen in the thoracic ganglion. A similar increment in surface expression of GluN1 in the central brain is observed 3 h after administration of the competitive GABAA receptor antagonist, bicuculline. These consolidation changes are part of a plasticity event that first, during the down-regulation, stabilizes the trace and later, at 3-h post-training, changes the threshold for synapse activation. PMID:27421895

  13. Quantitative measurement of the levels of melanocortin receptor subtype 1, 2, 3 and 5 and pro-opio-melanocortin peptide gene expression in subsets of human peripheral blood leucocytes.

    PubMed

    Andersen, G N; Hägglund, M; Nagaeva, O; Frängsmyr, L; Petrovska, R; Mincheva-Nilsson, L; Wikberg, J E S

    2005-03-01

    Levels of the melanocortin receptor (MCR) 1, 2, 3 and 5 subtypes and pro-opio-melanocortin (POMC) protein mRNA were measured by the real-time quantitative reverse transcriptase polymerase chain reaction method in CD4+ T helper (Th) cells, CD8+ T cytotoxic cells, CD19+ B cells, CD56+ natural killer (NK) cells, CD14+ monocytes and CD15+ granulocytes from healthy donors. We found high levels of all of the MC1, 2, 3 and 5R subtype mRNA in Th cells and moderate levels in NK cells, monocytes and granulocytes. POMC peptide mRNA was found in all examined leucocyte subsets, but only low levels were present in granulocytes. Our findings suggest a co-ordinating role for MCR subtypes and their naturally occurring ligands in the co-operation between innate and adaptive immunity. Moreover, our findings are compatible with earlier finding of MCR-mediated tolerance induction in Th cells. PMID:15787746

  14. Enhanced expression of G-protein coupled estrogen receptor (GPER/GPR30) in lung cancer

    PubMed Central

    2012-01-01

    Background G-protein-coupled estrogen receptor (GPER/GPR30) was reported to bind 17β-estradiol (E2), tamoxifen, and ICI 182,780 (fulvestrant) and promotes activation of epidermal growth factor receptor (EGFR)-mediated signaling in breast, endometrial and thyroid cancer cells. Although lung adenocarcinomas express estrogen receptors α and β (ERα and ERβ), the expression of GPER in lung cancer has not been investigated. The purpose of this study was to examine the expression of GPER in lung cancer. Methods The expression patterns of GPER in various lung cancer lines and lung tumors were investigated using standard quantitative real time PCR (at mRNA levels), Western blot and immunohistochemistry (IHC) methods (at protein levels). The expression of GPER was scored and the pairwise comparisons (cancer vs adjacent tissues as well as cancer vs normal lung tissues) were performed. Results Analysis by real-time PCR and Western blotting revealed a significantly higher expression of GPER at both mRNA and protein levels in human non small cell lung cancer cell (NSCLC) lines relative to immortalized normal lung bronchial epithelial cells (HBECs). The virally immortalized human small airway epithelial cell line HPL1D showed higher expression than HBECs and similar expression to NSCLC cells. Immunohistochemical analysis of tissue sections of murine lung adenomas as well as human lung adenocarcinomas, squamous cell carcinomas and non-small cell lung carcinomas showed consistently higher expression of GPER in the tumor relative to the surrounding non-tumor tissue. Conclusion The results from this study demonstrate increased GPER expression in lung cancer cells and tumors compared to normal lung. Further evaluation of the function and regulation of GPER will be necessary to determine if GPER is a marker of lung cancer progression. PMID:23273253

  15. Steroid Receptor Coactivator-2 Expression in Brain and Physical Associations with Steroid Receptors

    PubMed Central

    Yore, Mackensie A.; Im, DaEun; Webb, Lena K.; Zhao, Yingxin; Chadwick, Joseph G.; Molenda-Figueira, Heather A.; Haidacher, Sigmund J.; Denner, Larry; Tetel, Marc J.

    2010-01-01

    Estradiol and progesterone bind to their respective receptors in the hypothalamus and hippocampus to influence a variety of behavioral and physiological functions, including reproduction and cognition. Work from our lab and others has shown that the nuclear receptor coactivators, steroid receptor coactivator-1 (SRC-1) and SRC-2, are essential for efficient estrogen receptor (ER) and progestin receptor (PR) transcriptional activity in brain and for hormone-dependent behaviors. While the expression of SRC-1 in brain has been studied extensively, little is known about the expression of SRC-2 in brain. In the present studies, we found that SRC-2 was highly expressed throughout the hippocampus, amygdala and hypothalamus, including the medial preoptic area (MPOA), ventral medial nucleus (VMN), arcuate nucleus (ARC), bed nucleus of the stria terminalis, supraoptic nucleus and suprachiasmatic nucleus. In order for coactivators to function with steroid receptors, they must be expressed in the same cells. Indeed, SRC-2 and ERα were coexpressed in many cells in the MPOA, VMN and ARC, all brain regions known to be involved in female reproductive behavior and physiology. While in vitro studies indicate that SRC-2 physically associates with ER and PR, very little is known about receptor-coactivator interactions in brain. Therefore, we used pull-down assays to test the hypotheses that SRC-2 from hypothalamic and hippocampal tissue physically associate with ER and PR subtypes in a ligand-dependent manner. SRC-2 from both brain regions interacted with ERα bound to agonist, but not in the absence of ligand or in the presence of the selective ER modulator, tamoxifen. Analysis by mass spectrometry confirmed these ligand-dependent interactions between ERα and SRC-2 from brain. In dramatic contrast, SRC-2 from brain showed little to no interaction with ERβ. Interestingly, SRC-2 from both brain regions interacted with PR-B, but not PR-A, in a ligand-dependent manner. Taken together

  16. Two unrelated putative membrane-bound progestin receptors, progesterone membrane receptor component 1 (PGMRC1) and membrane progestin receptor (mPR) beta, are expressed in the rainbow trout oocyte and exhibit similar ovarian expression patterns

    PubMed Central

    Mourot, Brigitte; Nguyen, Thaovi; Fostier, Alexis; Bobe, Julien

    2006-01-01

    Background In lower vertebrates, steroid-induced oocyte maturation is considered to involve membrane-bound progestin receptors. Two totally distinct classes of putative membrane-bound progestin receptors have been reported in vertebrates. A first class of receptors, now termed progesterone membrane receptor component (PGMRC; subtypes 1 and 2) has been studied since 1996 but never studied in a fish species nor in the oocyte of any animal species. A second class of receptors, termed membrane progestin receptors (mPR; subtypes alpha, beta and gamma), was recently described in vertebrates and implicated in the progestin-initiated induction of oocyte maturation in fish. Methods In the present study, we report the characterization of the full coding sequence of rainbow trout PGMRC1 and mPR beta cDNAs, their tissue distribution, their ovarian expression profiles during oogenesis, their hormonal regulation in the full grown ovary and the in situ localization of PGMRC1 mRNA in the ovary. Results Our results clearly show, for the first time in any animal species, that rainbow trout PGMRC1 mRNA is present in the oocyte and has a strong expression in ovarian tissue. In addition, we show that both mPR beta and PGMRC1, two members of distinct membrane-bound progestin receptor classes, exhibit highly similar ovarian expression profiles during the reproductive cycle with maximum levels during vitellogenesis and a down-expression during late vitellogenesis. In addition, the mRNA abundance of both genes is not increased after in vitro hormonal stimulation of full grown follicles by maturation inducing hormones. Conclusion Together, our findings suggest that PGMRC1 is a new possible participant in the progestin-induced oocyte maturation in fish. However, its participation in the process of oocyte maturation, which remains to be confirmed, would occur at post-transcriptional levels. PMID:16457725

  17. High basal levels of functional toll-like receptor 3 (TLR3) and noncanonical Wnt5a are expressed in papillary thyroid cancer and are coordinately decreased by phenylmethimazole together with cell proliferation and migration.

    PubMed

    McCall, Kelly D; Harii, Norikazu; Lewis, Christopher J; Malgor, Ramiro; Kim, Won Bae; Saji, Motoyasu; Kohn, Aimee D; Moon, Randall T; Kohn, Leonard D

    2007-09-01

    High basal levels of TLR3 and Wnt5a RNA are present in papillary thyroid carcinoma (PTC) cell lines consistent with their overexpression and colocalization in PTC cells in vivo. This is not the case in thyrocytes from normal tissue and in follicular carcinoma (FC) or anaplastic carcinoma (AC) cells or tissues. The basally expressed TLR3 are functional in PTC cells as evidenced by the ability of double-strand RNA (polyinosine-polycytidylic acid) to significantly increase the activity of transfected NF-kappaB and IFN-beta luciferase reporter genes and the levels of two end products of TLR3 signaling, IFN-beta and CXCL10. Phenylmethimazole (C10), a drug that decreases TLR3 expression and signaling in FRTL-5 thyrocytes, decreases TLR3 levels and signaling in PTC cells in a concentration-dependent manner. C10 also decreased Wnt5a RNA levels coordinate with decreases in TLR3. E-cadherin RNA levels, whose suppression may be associated with high Wnt5a, increased with C10 treatment. C10 simultaneously decreased PTC proliferation and cell migration but had no effect on the growth and migration of FC, AC, or FRTL-5 cells. C10 decreases high basal phosphorylation of Tyr705 and Ser727 on Stat3 in PTC cells and inhibits IL-6-induced Stat3 phosphorylation. IL-6-induced Stat3 phosphorylation is important both in up-regulating Wnt5a levels and in cell growth. In sum, high Wnt5a levels in PTC cells may be related to high TLR3 levels and signaling; and the ability of phenylmethimazole (C10) to decrease growth and migration of PTC cells may be related to its suppressive effect on TLR3 and Wnt5a signaling, particularly Stat3 activation. PMID:17525119

  18. Perturbing the Cellular Levels of Steroid Receptor Coactivator-2 Impairs Murine Endometrial Function

    PubMed Central

    Szwarc, Maria M.; Kommagani, Ramakrishna; Jeong, Jae-Wook; Wu, San-Pin; Tsai, Sophia Y.; Tsai, Ming-Jer; O’Malley, Bert W.; DeMayo, Francesco J.; Lydon, John P.

    2014-01-01

    As pleiotropic coregulators, members of the p160/steroid receptor coactivator (SRC) family control a broad spectrum of transcriptional responses that underpin a diverse array of physiological and pathophysiological processes. Because of their potent coregulator properties, strict controls on SRC expression levels are required to maintain normal tissue functionality. Accordingly, an unwarranted increase in the cellular levels of SRC members has been causally linked to the initiation and/or progression of a number of clinical disorders. Although knockout mouse models have underscored the critical non-redundant roles for each SRC member in vivo, there are surprisingly few mouse models that have been engineered to overexpress SRCs. This deficiency is significant since SRC involvement in many of these disorders is based on unscheduled increases in the levels (rather than the absence) of SRC expression. To address this deficiency, we used recent mouse technology that allows for the targeted expression of human SRC-2 in cells which express the progesterone receptor. Through cre-loxP recombination driven by the endogenous progesterone receptor promoter, a marked elevation in expression levels of human SRC-2 was achieved in endometrial cells that are positive for the progesterone receptor. As a result of this increase in coregulator expression, female mice are severely subfertile due to a dysfunctional uterus, which exhibits a hypersensitivity to estrogen exposure. Our findings strongly support the proposal from clinical observations that increased levels of SRC-2 are causal for a number of endometrial disorders which compromise fertility. Future studies will use this mouse model to decipher the molecular mechanisms that underpin the endometrial defect. We believe such mechanistic insight may provide new molecular descriptors for diagnosis, prognosis, and/or therapy in the clinical management of female infertility. PMID:24905738

  19. Urokinase receptor is a multifunctional protein: influence of receptor occupancy on macrophage gene expression.

    PubMed Central

    Rao, N K; Shi, G P; Chapman, H A

    1995-01-01

    Binding of urokinase to the glycolipid-anchored urokinase receptor (uPAR) has been implicated in macrophage differentiation. However, no biochemical markers of differentiation have yet been directly linked to uPAR occupancy. As extensive changes in proteolytic profile characterize monocytic differentiation, we have examined the role of uPAR occupancy on protease expression by differentiating phagocytes. Antibodies to either urokinase or to uPAR that prevent receptor binding inhibited induction of cathepsin B in cultured monocytes and both cathepsin B and 92-kD gelatinase mRNA and protein in phorbol diester-stimulated myeloid cells. Mannosamine, an inhibitor of glycolipid anchor assembly, also blocked protease expression. Anti-catalytic urokinase antibodies, excess inactive urokinase, or aprotinin had no effect, indicating that receptor occupancy per se regulated protease expression. Antibodies to the integrins CD11a and CD29 or to the glycolipid-anchored proteins CD14 and CD55 also had no effect. Protease induction was independent of matrix attachment. Antibodies to urokinase or uPAR affected neither the decrease in cathepsin G nor the increase in tumor necrosis factor-alpha in phorbol ester-stimulated cells. These data establish that uPAR is a multifunctional receptor, not only promoting pericellular proteolysis and matrix attachment, but also effecting cysteine- and metallo-protease expression during macrophage differentiation. Images PMID:7615819

  20. Identification of Type II Interferon Receptors in Geese: Gene Structure, Phylogenetic Analysis, and Expression Patterns

    PubMed Central

    Zhou, Hao; Chen, Shun; Qi, Yulin; Zhou, Qin; Wang, Mingshu; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Liu, Fei; Chen, Xiaoyue; Cheng, Anchun

    2015-01-01

    Interferon γ receptor 1 (IFNGR1) and IFNGR2 are two cell membrane molecules belonging to class II cytokines, which play important roles in the IFN-mediated antiviral signaling pathway. Here, goose IFNGR1 and IFNGR2 were cloned and identified for the first time. Tissue distribution analysis revealed that relatively high levels of goose IFNγ mRNA transcripts were detected in immune tissues, including the harderian gland, cecal tonsil, cecum, and thymus. Relatively high expression levels of both IFNGR1 and IFNGR2 were detected in the cecal tonsil, which implicated an important role of IFNγ in the secondary immune system of geese. No specific correlation between IFNγ, IFNGR1, and IFNGR2 expression levels was observed in the same tissues of healthy geese. IFNγ and its cognate receptors showed different expression profiles, although they appeared to maintain a relatively balanced state. Furthermore, the agonist R848 led to the upregulation of goose IFNγ but did not affect the expression of goose IFNGR1 or IFNGR2. In summary, trends in expression of goose IFNγ and its cognate receptors showed tissue specificity, as well as an age-related dependency. These findings may help us to better understand the age-related susceptibility to pathogens in birds. PMID:26345454

  1. Cloning, expression and pharmacological characterization of rabbit adenosine A1 and A3 receptors.

    PubMed

    Hill, R J; Oleynek, J J; Hoth, C F; Kiron, M A; Weng, W; Wester, R T; Tracey, W R; Knight, D R; Buchholz, R A; Kennedy, S P

    1997-01-01

    The role of adenosine A1 and A3 receptors in mediating cardioprotection has been studied predominantly in rabbits, yet the pharmacological characteristics of rabbit adenosine A1 and A3 receptor subtypes are unknown. Thus, the rabbit adenosine A3 receptor was cloned and expressed, and its pharmacology was compared with that of cloned adenosine A1 receptors. Stable transfection of rabbit A1 or A3 cDNAs in Chinese hamster ovary-K1 cells resulted in high levels of expression of each of the receptors, as demonstrated by high-affinity binding of the A1/A3 adenosine receptor agonist N6-(4-amino-3-[125I]iodobenzyl)adenosine (125I-ABA). For both receptors, binding of 125I-ABA was inhibited by the GTP analog 5'-guanylimidodiphosphate, and forskolin-stimulated cyclic AMP accumulation was inhibited by the adenosine receptor agonist (R)-phenylisopropyladenosine. The rank orders of potency of adenosine receptor agonists for inhibition of 125I-ABA binding were as follows: rabbit A1, N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N-ethylcarboxamidoadenosine > or = I-ABA > or = N6-2-(4-aminophenyl) ethyladenosine > > N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > N6-(4-amino-3-benzyl)adenosine; rabbit A3, N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide > or = I-ABA > > N-ethylcarboxamidoadenosine > N6-2-(4-aminophenyl) ethyladenosine = N6-cyclopentyladenosine = (R)-phenylisopropyladenosine > N6-(4-amino-3-benzyl)adenosine. The adenosine receptor antagonist rank orders were as follow: rabbit A1, 8-cyclopentyl-1,3-dipropylxanthine > 1,3- dipropyl-8-(4-acrylate)phenylxanthine > or = xanthine amine congener > > 8-(p-sulfophenyl)theophylline; rabbit A3, xanthine amine congener > 1,3-dipropyl-8-(4-acrylate)phenylxanthine > or = 8-cyclopentyl-1,3-dipropylxanthine > > 8-(p-sulfophenyl)theophylline. These observations confirm the identity of the expressed proteins as A1 and A3 receptors. The results will facilitate further in-depth studies of the roles of A1 and A3 receptors in

  2. Prostaglandin E specifically upregulates the expression of the mannose-receptor on mouse bone marrow-derived macrophages.

    PubMed Central

    Schreiber, S; Blum, J S; Chappel, J C; Stenson, W F; Stahl, P D; Teitelbaum, S L; Perkins, S L

    1990-01-01

    The macrophage mannose receptor (MMR) facilitates the binding and internalization of microorganisms and glycoproteins with terminal mannose residues. The receptor is progressively upregulated as bone marrow precursor cells mature into macrophages and thus may serve as a marker of differentiation. Prostaglandins of the E series (PGE) are known inhibitors of monocyte and macrophage precursor proliferation, an effect often associated with cellular maturation. MMR expression was therefore assessed after exposure of bone marrow macrophage precursor (BMMP) cells to these prostanoids. Receptor expression was determined by ligand binding and via immunoprecipitation of newly synthesized receptor molecules. PGE1 and PGE2 at 10(-9)-10(-6) M upregulated MMR surface expression and biosynthesis four- to sixfold in a dose-dependent manner. BMMPs responsive to prostaglandins were characterized by plastic adherence, F4/80 antigen expression, and nonspecific esterase activity. Prostaglandins accelerated the expression of the MMR in cells by 48-72h, with maximal levels of receptor expression being identical in control or treated cells. Thus, prostaglandins enhanced mannose receptor expression in adherent but not fully differentiated macrophage precursors. This effect is specific for PGE and is mimicked by dibutyrl cyclic AMP. These results indicate that prostaglandins accelerate MMR expression and hence the differentiation of macrophage precursor cells. Cells resident in the bone marrow secrete abundant prostaglandins, suggesting that a paracrine mechanism may exist to regulate MMR expression and function. Images PMID:1965946

  3. mRNA expression pattern of gonadotropin receptors in bovine follicular cysts.

    PubMed

    Marelli, Belkis E; Diaz, Pablo U; Salvetti, Natalia R; Rey, Florencia; Ortega, Hugo H

    2014-12-01

    Follicular growth and steroidogenesis are dependent on gonadotropin binding to their receptors in granulosa and theca cells of ovarian follicles. The aim of the present study was to evaluate the expression patterns of follicle-stimulating hormone receptor (FSHR) and luteinizing hormone receptor (LHCGR) in ovarian follicular structures from cows with cystic ovarian disease (COD) as compared with those of regularly cycling cows. Relative real-time RT-PCR analysis showed that the expression of FSHR mRNA in granulosa cells was highest in small antral follicles, then decreased significantly as follicles increased in size, and was lowest in cysts. FSHR mRNA was not detected in the theca cells of any follicular category, including cysts. LHCGR mRNA expression in granulosa cells was significantly higher in large antral follicles than in cysts, and not detected in granulosa cells of small and medium antral follicles. In theca cells, the expression level of LHCGR mRNA in medium antral follicles was higher than in small and large antral follicles, whereas that in follicular cysts it was similar to those in small and medium antral follicles, but higher than that in large antral follicles. Our findings provide evidence that there is an altered gonadotropin receptor expression in bovine cystic follicles, and suggest that in conditions characterized by altered ovulation, such as COD, changes in the signaling system of gonadotropins may play a fundamental role in their pathogenesis. PMID:25454493

  4. Expressions of lysophosphatidic acid receptors in the development of human ovarian carcinoma

    PubMed Central

    Si, Jinge; Su, Yuanyuan; Wang, Yifeng; Yan, You-Liang; Tang, Ya-Ling

    2015-01-01

    Aim: To investigate the associations between the expressions of three lysophosphatidic acid (LPA) receptors (LPA1-3) and the development of ovarian carcinoma (OC). Method: Ovarian tissue specimens, including normal ovarian epithelium tissues, benign ovarian tumor tissues and OC tissues were collected from patients who underwent surgical resections between March 2012 and December 2014. Immunohistochemical staining was used to detect LPA receptor expressions in ovarian tissues. Reverse transcription-polymerase chain reaction and Western blotting were used to detect mRNA and protein expression of LPA receptors, respectively. Association analysis between LPA receptors protein expression and clinical pathological characteristics was conducted. The value of LPA2 and LPA3 in discriminating OC was confirmed by receiver-operator characteristic (ROC) curves analysis. Results: The positive expression rates of LPA2 and LPA3 in OC group was obviously higher than normal control and benign groups. The LPA2 and LPA3 mRNA and protein levels in OC group were higher than in normal control and benign groups. LPA2 and LPA3 mRNA expression levels were positively correlated with LPA2 and LPA3 protein expression in OC group. ROC curve analysis revealed that LPA2 yield a specificity of 96.3% and a sensitivity of 97.9%, and LPA3 yield a specificity of 98.5% and a sensitivity of 97.9% for the detection of OC. Conclusion: LPA2 and LPA3 were highly expressed in OC tissues, which may be involved in the development of OC. Further, LPA2 and LPA3 had higher sensitivity and specificity in distinguishing the OC from benign ovarian tumors, which could be potential diagnostic indictors in OC. PMID:26770382

  5. Prefrontal GABA(A) receptor alpha-subunit expression in normal postnatal human development and schizophrenia.

    PubMed

    Duncan, Carlotta E; Webster, Maree J; Rothmond, Debora A; Bahn, Sabine; Elashoff, Michael; Shannon Weickert, Cynthia

    2010-07-01

    Cortical GABA deficits that are consistently reported in schizophrenia may reflect an etiology of failed normal postnatal neurotransmitter maturation. Previous studies have found prefrontal cortical GABA(A) receptor alpha subunit alterations in schizophrenia, yet their relationship to normal developmental expression profiles in the human cortex has not been determined. The aim of this study was to quantify GABA(A) receptor alpha-subunit mRNA expression patterns in human dorsolateral prefrontal cortex (DLPFC) during normal postnatal development and in schizophrenia cases compared to controls. Transcript levels of GABA(A) receptor alpha subunits were measured using microarray and qPCR analysis of 60 normal individuals aged 6weeks to 49years and in 37 patients with schizophrenia/schizoaffective disorder and 37 matched controls. We detected robust opposing changes in cortical GABA(A) receptor alpha1 and alpha5 subunits during the first few years of postnatal development, with a 60% decrease in alpha5 mRNA expression and a doubling of alpha1 mRNA expression with increasing age. In our Australian schizophrenia cohort we detected decreased GAD67 mRNA expression (p=0.0012) and decreased alpha5 mRNA expression (p=0.038) in the DLPFC with no significant change of other alpha subunits. Our findings confirm that GABA deficits (reduced GAD67) are a consistent feature of schizophrenia postmortem brain studies. Our study does not confirm alterations in cortical alpha1 or alpha2 mRNA levels in the schizophrenic DLPFC, as seen in previous studies, but instead we report a novel down-regulation of alpha5 subunit mRNA suggesting that post-synaptic alterations of inhibitory receptors are an important feature of schizophrenia but may vary between cohorts. PMID:20100621

  6. Decreased expression of thyroid receptor-associated protein 220 in temporal lobe tissue of patients with refractory epilepsy

    SciTech Connect

    Li Jinmei . E-mail: jinmeimery@Yahoo.com.cn; Wang Xuefeng . E-mail: rengang68@vip.sina.com; Xi Zhiqin; Gong Yun; Liu Fengying; Sun Jijun; Wu Yuan; Luan Guoming; Wang Yuping; Li Yunlin; Zhang Jianguo; Lu Yong; Li Hongwei

    2006-10-06

    Purpose: TRAP220 (thyroid hormone receptor-associated protein) functions as a coactivator for nuclear receptors and stimulates transcription by recruiting the TRAP mediator complex to hormone responsive promoter regions. Thus, TRAP220 enhances the function of thyroid/steroid hormone receptors such as thyroid hormone and oestrogen receptors. This study investigated the expression of TRAP220 mRNA and protein level in epileptic brains comparing with human control. Methods: We examined the expression of TRAP220 mRNA and protein levels in temporal lobes from patients with chronic pharmacoresistant epilepsy who have undergone surgery. Results: Expression of TRAP220 mRNA and protein was shown to be decreased significantly in the temporal cortex of the patients with epilepsy. Conclusions: Our work showed that a decrease in TRAP220 mRNA and protein levels may be involved in the pathophysiology of epilepsy and may be associated with impairment of the brain caused by frequent seizures.

  7. Increased hypothalamic 5-HT2A receptor gene expression and effects of pharmacologic 5-HT2A receptor inactivation in obese A{sup y} mice

    SciTech Connect

    Nonogaki, Katsunori . E-mail: knonogaki-tky@umin.ac.jp; Nozue, Kana; Oka, Yoshitomo

    2006-12-29

    Serotonin (5-hydroxytryptamine; 5-HT) 2A receptors contribute to the effects of 5-HT on platelet aggregation and vascular smooth muscle cell proliferation, and are reportedly involved in decreases in plasma levels of adiponectin, an adipokine, in diabetic subjects. Here, we report that systemic administration of sarpogrelate, a 5-HT2A receptor antagonist, suppressed appetite and increased hypothalamic pro-opiomelanocortin and cocaine- and amphetamine-regulated transcript, corticotropin releasing hormone, 5-HT2C, and 5-HT1B receptor gene expression. A{sup y} mice, which have ectopic expression of the agouti protein, significantly increased hypothalamic 5-HT2A receptor gene expression in association with obesity compared with wild-type mice matched for age. Systemic administration of sarpogrelate suppressed overfeeding, body weight gain, and hyperglycemia in obese A{sup y} mice, whereas it did not increase plasma adiponectin levels. These results suggest that obesity increases hypothalamic 5-HT2A receptor gene expression, and pharmacologic inactivation of 5-HT2A receptors inhibits overfeeding and obesity in A{sup y} mice, but did not increase plasma adiponectin levels.

  8. Influence of minor thermal injury on expression of complement receptor CR3 on human neutrophils.

    PubMed Central

    Nelson, R. D.; Hasslen, S. R.; Ahrenholz, D. H.; Haus, E.; Solem, L. D.

    1986-01-01

    Thermal injury is well known to inhibit functions of the circulating neutrophil related to its role in host defense against infection, but the mechanism(s) of this phenomenon are not fully understood. To gain further clues to these mechanisms, the authors have studied patients with thermal injury in terms of altered expression of neutrophil cell membrane receptors for the opsonic complement-derived ligand C3bi--complement receptor Type 3, or CR3. CR3 expression was selected for study because an increase in the number of receptors on the cell surface can be stimulated by products of complement activation known to accumulate after thermal injury and because of the role of CR3 in phagocytic and adherence functions of the neutrophil. Expression of CR3 was monitored semiquantitatively by flow cytometry with the use of a murine monoclonal antibody (OKM1) specific for an antigen (CD11) associated with this receptor. Patients evaluated were limited in this study to those with minor degrees of thermal injury (second-degree burn involving less than 20% of total body surface area) so that possible confounding effects of major injury and its complications could be eliminated. It was observed that patient neutrophil CR3 becomes significantly up-regulated during the first week, as early as 1 day after injury. The maximum level of expression of CR3 averaged greater than 150% (range, 70-314%) of the respective minimum level observed for each patient. The minimum levels of expression of CR3 on patient neutrophils, reached 11-37 days after injury for 7 of 8 patients, were comparable to the level of expression of CR3 on unstimulated control neutrophils. Such temporal up-regulation of patient neutrophil CR3 suggests the early generation of stimuli of CR3 mobilization in response to thermal injury. Increased numbers of CR3 on patient neutrophils may augment microbicidal function and enhance or inhibit delivery of cells to the burn site. PMID:3541642

  9. Translation Levels Control Multi-Spanning Membrane Protein Expression

    PubMed Central

    Brown, Cecilia; Bostrom, Jenny; Fuh, Germaine; Lee, Chingwei V.; Huang, Arthur; Vandlen, Richard L.; Yansura, Daniel G.

    2012-01-01

    Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane. PMID:22563408

  10. Expression of Tachykinins and Tachykinin Receptors and Interaction with Kisspeptin in Human Granulosa and Cumulus Cells.

    PubMed

    García-Ortega, Jordán; Pinto, Francisco M; Prados, Nicolás; Bello, Aixa R; Almeida, Teresa A; Fernández-Sánchez, Manuel; Candenas, Luz

    2016-06-01

    The neurokinin B/NK3 receptor (NK3R) and kisspeptin/kisspeptin receptor (KISS1R), two systems which are essential for reproduction, are coexpressed in human mural granulosa (MGC) and cumulus cells (CCs). However, little is known about the presence of other members of the tachykinin family in the human ovary. In the present study, we analyzed the expression of substance P (SP), hemokinin-1 (HK-1), NK1 receptor (NK1R), and NK2 receptor (NK2R) in MGCs and CCs collected from preovulatory follicles of oocyte donors at the time of oocyte retrieval. RT-PCR, quantitative RT-PCR, immunocytochemistry, and Western blotting were used to investigate the patterns of expression of tachykinin and tachykinin receptor mRNAs and proteins and the possible interaction between the tachykinin family and kisspeptin. Intracellular free Ca(2+) levels ([Ca(2+)]i) in MGCs after exposure to SP or kisspeptin in the presence of SP were also measured. We found that SP, HK-1, the truncated NK1R isoform NK1R-Tr, and NK2R were all expressed in MGCs and CCs. NK1R-Tr mRNA and NK2R mRNA and protein levels were higher in MGCs than in CCs from the same patients. Treatment of cells with kisspeptin modulated the expression of HK-1, NK3R, and KISS1R mRNAs, whereas treatment with SP regulated kisspeptin mRNA levels and reduced the [Ca(2+)]i response produced by kisspeptin. These data demonstrate that the whole tachykinin system is expressed and acts in coordination with kisspeptin to regulate granulosa cell function in the human ovary. PMID:27146034

  11. Dopamine inhibits somatolactin gene expression in tilapia pituitary cells through the dopamine D2 receptors.

    PubMed

    Jiang, Quan; Lian, Anji; He, Qi

    2016-07-01

    Dopamine (DA) is an important neurotransmitter in the central nervous system of vertebrates and possesses key hypophysiotropic functions. Early studies have shown that DA has a potent inhibitory effect on somatolactin (SL) release in fish. However, the mechanisms responsible for DA inhibition of SL gene expression are largely unknown. To this end, tilapia DA type-1 (D1) and type-2 (D2) receptor transcripts were examined in the neurointermediate lobe (NIL) of the tilapia pituitary by real-time PCR. In tilapia, DA not only was effective in inhibiting SL mRNA levels in vivo and in vitro, but also could abolish pituitary adenylate cyclase-activating polypeptide (PACAP)- and salmon gonadotropin-releasing hormone (sGnRH)-stimulated SL gene expression at the pituitary level. In parallel studies, the specific D2 receptor agonists quinpirole and bromocriptine could mimic the DA-inhibited SL gene expression. Furthermore, the D2 receptor antagonists domperidone and (-)-sulpiride could abolish the SL response to DA or the D2 agonist quinpirole, whereas D1 receptor antagonists SCH23390 and SKF83566 were not effective in this respect. In primary cultures of tilapia NIL cells, D2 agonist quinpirole-inhibited cAMP production could be blocked by co-treatment with the D2 antagonist domperidone and the ability of forskolin to increase cAMP production was also inhibited by quinpirole. Using a pharmacological approach, the AC/cAMP pathway was shown to be involved in quinpirole-inhibited SL mRNA expression. These results provide evidence that DA can directly inhibit SL gene expression at the tilapia pituitary level via D2 receptor through the AC/cAMP-dependent mechanism. PMID:26970582

  12. Molecular characterization of a mouse prostaglandin D receptor and functional expression of the cloned gene.

    PubMed

    Hirata, M; Kakizuka, A; Aizawa, M; Ushikubi, F; Narumiya, S

    1994-11-01

    Prostanoid receptors belong to the family of G protein-coupled receptors with seven transmembrane domains. By taking advantage of nucleotide sequence homology among the prostanoid receptors, we have isolated and identified a cDNA fragment and its gene encoding a mouse prostaglandin (PG) D receptor by reverse transcription polymerase chain reaction and gene cloning. This gene codes for a polypeptide of 357 amino acids, with a calculated molecular weight of 40,012. The deduced amino acid sequence has a high degree of similarity with the mouse PGI receptor and the EP2 subtype of the PGE receptor, which together form a subgroup of the prostanoid receptors. Chinese hamster ovary cells stably expressing the gene showed a single class of binding sites for [#H]PGD2 with a Kd of 40 nM. This binding was displaced by unlabeled ligands in the following order: PGD2 > BW 245C (a PGD agonist) > BW A868C (a PGD antagonist) > STA2 (a thromboxane A2 agonist). PGE2, PGF2 alpha, and iloprost showed little displacement activity at concentrations up to 10 microM. PGD2 and BW 245C also increased cAMP levels in Chinese hamster ovary cells expressing the receptor, in a concentration-dependent manner. BW A868C showed a partial agonist activity in the cAMP assay. Northern blotting analysis with mouse poly(A)+ RNA identified a major mRNA species of 3.5 kb that was most abundantly expressed in the ileum, followed by lung, stomach, and uterus. PMID:7972033

  13. Inflammatory Eicosanoids Increase Amyloid Precursor Protein Expression via Activation of Multiple Neuronal Receptors

    PubMed Central

    Herbst-Robinson, Katie J.; Liu, Li; James, Michael; Yao, Yuemang; Xie, Sharon X.; Brunden, Kurt R.

    2015-01-01

    Senile plaques comprised of Aβ peptides are a hallmark of Alzheimer’s disease (AD) brain, as are activated glia that release inflammatory molecules, including eicosanoids. Previous studies have demonstrated that amyloid precursor protein (APP) and Aβ levels can be increased through activation of thromboxane A2-prostanoid (TP) receptors on neurons. We demonstrate that TP receptor regulation of APP expression depends on Gαq-signaling and conventional protein kinase C isoforms. Importantly, we discovered that Gαq-linked prostaglandin E2 and leukotriene D4 receptors also regulate APP expression. Prostaglandin E2 and thromboxane A2, as well as total APP levels, were found to be elevated in the brains of aged 5XFAD transgenic mice harboring Aβ plaques and activated glia, suggesting that increased APP expression resulted from eicosanoid binding to Gαq-linked neuronal receptors. Notably, inhibition of eicosanoid synthesis significantly lowered brain APP protein levels in aged 5XFAD mice. These results provide new insights into potential AD therapeutic strategies. PMID:26672557

  14. Prefrontal cortical-striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity.

    PubMed

    Simon, Nicholas W; Beas, Blanca S; Montgomery, Karienn S; Haberman, Rebecca P; Bizon, Jennifer L; Setlow, Barry

    2013-06-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  15. Prefrontal cortical–striatal dopamine receptor mRNA expression predicts distinct forms of impulsivity

    PubMed Central

    Simon, Nicholas W.; Beas, Blanca S.; Montgomery, Karienn S.; Haberman, Rebecca P.; Bizon, Jennifer L.; Setlow, Barry

    2014-01-01

    Variation in dopamine receptor levels has been associated with different facets of impulsivity. To further delineate the neural substrates underlying impulsive action (inability to withhold a prepotent motor response) and impulsive choice (delay aversion), we characterised rats in the Differential Reinforcement of Low Rates of Responding task and a delay discounting task. We also measured performance on an effort-based discounting task. We then assessed D1 and D2 dopamine receptor mRNA expression in subregions of the prefrontal cortex and nucleus accumbens using in situ hybridisation, and compared these data with behavioral performance. Expression of D1 and D2 receptor mRNA in distinct brain regions was predictive of impulsive action. A dissociation within the nucleus accumbens was observed between subregions and receptor subtypes; higher D1 mRNA expression in the shell predicted greater impulsive action, whereas lower D2 mRNA expression in the core predicted greater impulsive action. We also observed a negative correlation between impulsive action and D2 mRNA expression in the prelimbic cortex. Interestingly, a similar relationship was present between impulsive choice and prelimbic cortex D2 mRNA, despite the fact that behavioral indices of impulsive action and impulsive choice were uncorrelated. Finally, we found that both high D1 mRNA expression in the insular cortex and low D2 mRNA expression in the infralimbic cortex were associated with willingness to exert effort for rewards. Notably, dopamine receptor mRNA in these regions was not associated with either facet of impulsivity. The data presented here provide novel molecular and neuroanatomical distinctions between different forms of impulsivity, as well as effort-based decision-making. PMID:23510331

  16. The role of cannabinoid 1 receptor expressing interneurons in behavior

    PubMed Central

    Brown, Jacquelyn A.; Horváth, Szatmár; Garbett, Krassimira; Schmidt, Martin J.; Everheart, Monika; Gellért, Levente; Ebert, Philip; Mirnics, Károly

    2013-01-01

    Schizophrenia is a devastating neurodevelopmental disorder that affects approximately 1% of the population. Reduced expression of the 67-kD a protein isoform of glutamic acid decarboxylase (GAD67), is a hallmark of the disease, and is encoded by the GAD1 gene. In schizophrenia, GAD67 downregulation occurs in multiple interneuronal subpopulations, including the cannabinoid receptor type 1 positive (CNR1+) cells, but the functional consequences of these disturbances are not well understood. To investigate the role of the CNR1-positive GABA-ergic interneurons in behavioral and molecular processes, we employed a novel, miRNA-mediated transgenic mouse approach. We silenced the Gad1 transcript using a miRNA engineered to specifically target Gad1 mRNA under the control of Cnr1 bacterial artificial chromosome. Behavioral characterization of our transgenic mice showed elevated and persistent conditioned fear associated with an auditory cue and a significantly altered response to an amphetamine challenge. These deficits could not be attributed to sensory deficits or changes in baseline learning and memory. Furthermore, HPLC analyses revealed that Cnr1/Gad1 mice have enhanced serotonin levels, but not dopamine levels in response to amphetamine. Our findings demonstrate that dysfunction of a small subset of interneurons can have a profound effect on behavior and that the GABA-ergic, monoamine, and cannabinoid systems are functionally interconnected. The results also suggest that understanding the function of various interneuronal subclasses might be essential to develop knowledge-based treatment strategies for various mental disorders including schizophrenia and substance abuse. PMID:24239560

  17. Evidence of a correlation of estrogen receptor level and avian osteoclast estrogen responsiveness.

    PubMed

    Pederson, L; Kremer, M; Foged, N T; Winding, B; Ritchie, C; Fitzpatrick, L A; Oursler, M J

    1997-05-01

    Isolated osteoclasts from 5-week-old chickens respond to estradiol treatment in vitro with decreased resorption activity, increased nuclear proto-oncogene expression, and decreased lysosomal enzyme secretion. This study examines osteoclasts from embryonic chickens and egg-laying hens for evidence of estrogen responsiveness. Although osteoclasts from both of these sources express estrogen receptor mRNA and protein, estradiol treatment had no effect on resorption activity. In contrast to the lack of effect on resorption, estradiol treatment for 30 minutes resulted in steady-state mRNA levels of c-fos and c-jun increasing in osteoclasts from embryonic chickens and decreasing in osteoclasts from egg-laying hens. These data suggest that a nuclear proto-oncogene response may not be involved in estradiol-mediated decreased osteoclast resorption activity. To examine the influence of circulating estrogen on osteoclast estrogen responsiveness, 5-week-old chickens were injected with estrogen for 4 days prior to sacrifice. Estradiol treatment of osteoclasts from these chickens did not decrease resorption activity in vitro. Transfection of an estrogen receptor expression vector into osteoclasts from the estradiol-injected chickens and egg-laying hens restored estrogen responsiveness. Osteoclasts from 5-week-old chickens and estradiol treated 5-week-old chickens transfected with the estrogen receptor expression vector contained significantly higher levels of estrogen receptor protein and responded to estradiol treatment by decreasing secretion of cathepsins B and L and tartrate-resistant acid phosphatase. In contrast, osteoclasts from embryonic chickens, egg-laying hens, and estradiol-treated 5-week-old chickens either untransfected or transfected with an empty expression vector did not respond similarly. These data suggest that modulation of osteoclast estrogen responsiveness may be controlled by changes in the osteoclast estrogen receptor levels. PMID:9144340

  18. Bile acids inhibit duodenal secretin expression via orphan nuclear receptor small heterodimer partner (SHP).

    PubMed

    Lam, Ian P Y; Lee, Leo T O; Choi, Hueng-Sik; Alpini, Gianfranco; Chow, Billy K C

    2009-07-01

    Small heterodimer partner (SHP) is an orphan nuclear receptor in which gene expression can be upregulated by bile acids. It regulates its target genes by repressing the transcriptional activities of other nuclear receptors including NeuroD, which has been shown to regulate secretin gene expression. Here, we evaluated the regulation on duodenal secretin gene expression by SHP and selected bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA). In vitro treatment of CDCA or fexaramine elevated the SHP transcript level and occupancy on secretin promoter. The increase in the SHP level, induced by bile acid treatment or overexpression, reduced secretin gene expression, whereas this gene inhibitory effect was reversed by silencing of endogenous SHP. In in vivo studies, double-immunofluorescence staining demonstrated the coexpression of secretin and SHP in mouse duodenum. Feeding mice with 1% CA-enriched rodent chow resulted in upregulation of SHP and a concomitant decrease in secretin transcript and protein levels in duodenum compared with the control group fed with normal chow. A diet enriched with 5% cholestyramine led to a decrease in SHP level and a corresponding increase in secretin expression. Overall, this study showed that bile acids via SHP inhibit duodenal secretin gene expression. Because secretin is a key hormone that stimulates bile flow in cholangiocytes, this pathway thus provides a novel means to modulate secretin-stimulated choleresis in response to intraduodenal bile acids. PMID:19372104

  19. Depressed expression of Klotho and FGF receptor 1 in hyperplastic parathyroid glands from uremic patients.

    PubMed

    Komaba, Hirotaka; Goto, Shunsuke; Fujii, Hideki; Hamada, Yasuhiro; Kobayashi, Akira; Shibuya, Koji; Tominaga, Yoshihiro; Otsuki, Naoki; Nibu, Ken-Ichi; Nakagawa, Kimie; Tsugawa, Naoko; Okano, Toshio; Kitazawa, Riko; Fukagawa, Masafumi; Kita, Tomoyuki

    2010-02-01

    Fibroblast growth factor 23 (FGF23) exerts its effect by binding to its cognate FGF receptor 1 (FGFR1) in the presence of its co-receptor Klotho. Parathyroid glands express both FGFR1 and Klotho, and FGF23 decreases parathyroid hormone gene expression and hormone secretion directly. In uremic patients with secondary hyperparathyroidism (SHPT), however, parathyroid hormone secretion remains elevated despite extremely high FGF23 levels. To determine the mechanism of this resistance, we measured the expression of Klotho, FGFR1, and the proliferative marker Ki67 in 7 normal and 80 hyperplastic parathyroid glands from uremic patients by immunohistochemistry. All uremic patients had severe SHPT along with markedly high FGF23 levels. Quantitative real-time reverse transcription PCR showed that the mRNA levels for Klotho and FGFR1correlated significantly with their semi-quantitative immunohistochemical intensity. Compared with normal tissue, the immunohistochemical expression of Klotho and FGFR1 decreased, but Ki67 expression increased significantly in hyperplastic parathyroid glands, particularly in glands with nodular hyperplasia. These results suggest that the depressed expression of the Klotho-FGFR1 complex in hyperplastic glands underlies the pathogenesis of SHPT and its resistance to extremely high FGF23 levels in uremic patients. PMID:19890272

  20. Expression and plasticity of glutamate receptors in the supraoptic nucleus of the hypothalamus.

    PubMed

    Pak, C Wook; Currás-Collazo, Margarita C

    2002-01-15

    Magnocellular neuroendocrine cells (MNCs) of the supraoptic nucleus of the hypothalamus (SON) produce and release the hormones vasopressin (VP) and oxytocin (OT) in response to a variety of stimuli to regulate body water and salt, parturition and lactation. Hormone release is influenced by the pattern of neuronal firing of these MNCs, which, in turn, is governed by intrinsic conductances and synaptic inputs, including those mediated by the neurotransmitter glutamate. Functional and molecular evidence has confirmed the expression of AMPA-, NMDA-, and metabotropic-type glutamate receptors in the SON, that together may orchestrate the effects of glutamatergic transmission on neuroendocrine function. However, the specific roles of the different subtypes of glutamate receptors is not yet clear. As with other central neurons, the subunit composition of glutamate receptors on MNCs will likely determine their properties and may potentially help define the differential properties of VP- and OT-producing MNCs. Possible functions of glutamate receptors on SON MNCs include altering excitatory synaptic transmission of osmotic information, neuronal firing, hormone production and release, and calcium signaling. Of interest are the anatomical, molecular, and functional changes at glutamatergic synapses in the SON that occur in response to pertinent physiological stimuli or development. These types of plasticity may include changes in glutamatergic synaptic density, glutamate receptor levels, or glutamate receptor subunit expression, all of which can affect the efficiency of synaptic transmission. PMID:11810712

  1. Abnormally increased surface expression of AMPA receptors in the cerebellum, cortex and striatum of Cln3(-/-) mice.

    PubMed

    Kovács, Attila D; Hof, Caitlin; Pearce, David A

    2015-10-21

    Mutations in the CLN3 gene cause a fatal neurodegenerative disorder, juvenile CLN3 disease. Exploring the cause of the motor coordination deficit in the Cln3(-/-) mouse model of the disease we have previously found that attenuation of AMPA receptor activity in 1-month-old Cln3(-/-) mice significantly improves their motor coordination [20]. To elucidate the mechanism of the abnormally increased AMPA receptor function in Cln3(-/-) mice, we examined the surface expression of AMPA receptors using surface cross-linking in brain slices from 1-month-old wild type (WT) and Cln3(-/-) mice. In surface cross-linked brain samples, Western blotting for AMPA receptor subunits revealed significantly increased surface levels of GluA1 and GluA2 in the cerebellum, and of GluA2 in the cortex and striatum of Cln3(-/-) mice as compared to WT mice. Expression levels of the GluA4 subunit were similar in the cerebellum of WT and Cln3(-/-) mice. While intracellular GluA1 levels in the WT and Cln3(-/-) cerebellum or cortex were similar, the intracellular expression of GluA1 in the Cln3(-/-) striatum was decreased to 56% of the WT level. Our results show a prominent increase in AMPA receptor surface expression in the brain of Cln3(-/-) mice and suggest that CLN3 is involved in the regulation of AMPA receptor surface expression. PMID:26375929

  2. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines

    PubMed Central

    Hajiahmadi, S.; Panjehpour, M.; Aghaei, M.; Mousavi, S.

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  3. Molecular expression of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 human ovarian cancer cell lines.

    PubMed

    Hajiahmadi, S; Panjehpour, M; Aghaei, M; Mousavi, S

    2015-01-01

    Adenosine receptors (A1, A2a, A2b and A3) have several physiological and pathological roles in cancer cell lines. The present study was carried out to evaluate the mRNA and protein expression profile and functional role of adenosine receptors in OVCAR-3, Caov-4 and SKOV-3 ovarian cancer cell lines. The levels of mRNA and protein expression of A1, A2a, A2b and A3 adenosine receptors in the ovarian cancer cell lines were measured by Real-time PCR and western blotting. The functional roles of adenosine receptors were investigated through measurement of cAMP levels after agonist treatment. The mRNA and protein of all adenosine receptors subtypes were expressed in the ovarian cancer cell lines. Our findings demonstrated that A2b and A3 had the most mRNA and protein expression. Moreover, cAMP assay confirmed the functional role of A2b and A3 adenosine receptors. This findings demonstrated that A2b and A3 subtypes are most important adenosine receptors in humn ovarian cancer cell lines. This information provide a strong possibility into the relationship of A2b and A3 adenosine receptor and ovarian cancer. PMID:26430456

  4. The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

    PubMed

    Stoebner, P E; Carayon, P; Penarier, G; Fréchin, N; Barnéon, G; Casellas, P; Cano, J P; Meynadier, J; Meunier, L

    1999-06-01

    The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure. PMID:10354064

  5. GABAA Receptor Expression in the Forebrain of Ataxic Rolling Nagoya Mice.

    PubMed

    Nielsen, Elsebet Østergaard; Kaja, Simon

    2014-01-01

    The human CACNA1A gene encodes the pore-forming α1 subunit of CaV2.1 (P/Q-type) calcium channels and is the locus for several neurological disorders, including episodic ataxia type 2 (EA2), spinocerebellar ataxia type 6 (SCA6) and Familial Hemiplegic Migraine type 1 (FHM1). Several spontaneous mouse Cacna1a mutant strains exist, among them Rolling Nagoya (tg (rol)), carrying the R1262G point mutation in the mouse Cacna1a gene. tg (rol) mice display a phenotype of severe gait ataxia and motor dysfunction of the hind limbs. At the functional level, the R1262G mutation results in a positive shift of the activation voltage of the CaV2.1 channel and reduced current density. γ-Aminobutyric acid type A (GABAA) receptor subunit expression depends critically on neuronal calcium influx, and GABAA receptor dysfunction has previously been described for the cerebellum of tg (rol) and other ataxic Cacna1a mutant mice. Given the expression pattern of CaV2.1, it was hypothesized that calcium dysregulation in tg (rol) might affect GABAA receptor expression in the forebrain. Herein, functional GABAA receptors in the forebrain of tg (rol) mice were quantified and pharmacologically dissociated using [(3)H] radioligand binding. No gross changes to functional GABAA receptors were identified. Future cell type-specific analyses are required to identify possible cortical contributions to the psychomotor phenotype of tg (rol) mice. PMID:25309056

  6. Expression of endothelial cell-specific receptor tyrosine kinases and growth factors in human brain tumors.

    PubMed Central

    Hatva, E.; Kaipainen, A.; Mentula, P.; Jääskeläinen, J.; Paetau, A.; Haltia, M.; Alitalo, K.

    1995-01-01

    Key growth factor-receptor interactions involved in angiogenesis are possible targets for therapy of CNS tumors. Vascular endothelial growth factor (VEGF) is a highly specific endothelial cell mitogen that has been shown to stimulate angiogenesis, a requirement for solid tumor growth. The expression of VEGF, the closely related placental growth factor (PIGF), the newly cloned endothelial high affinity VEGF receptors KDR and FLT1, and the endothelial orphan receptors FLT4 and Tie were analyzed by in situ hybridization in normal human brain tissue and in the following CNS tumors: gliomas, grades II, III, IV; meningiomas, grades I and II; and melanoma metastases to the cerebrum. VEGF mRNA was up-regulated in the majority of low grade tumors studied and was highly expressed in cells of malignant gliomas. Significantly elevated levels of Tie, KDR, and FLT1 mRNAs, but not FLT4 mRNA, were observed in malignant tumor endothelia, as well as in endothelia of tissues directly adjacent to the tumor margin. In comparison, there was little or no receptor expression in normal brain vasculature. Our results are consistent with the hypothesis that these endothelial receptors are induced during tumor progression and may play a role in tumor angiogenesis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7856749

  7. Distribution of angiotensin type-1 receptor messenger RNA expression in the adult rat brain.

    PubMed

    Lenkei, Z; Palkovits, M; Corvol, P; Llorens-Cortes, C

    1998-02-01

    Angiotensin II and angiotensin III in the brain exert their various effects by acting on two pharmacologically well-defined receptors, the type-1 (AT1) and the type-2 (AT2) receptors. Receptor binding autoradiography has revealed the dominant presence of AT1 in brain nuclei involved in cardiovascular, body fluid and neuroendocrine control. The cloning of the AT1 complementary DNA has revealed the existence of two receptor subtypes in rodents, AT1A and AT1B. Using specific riboprobes for in situ hybridization, we have previously shown that the AT1A messenger RNA is predominantly expressed in the rat forebrain; in contrast the AT1B subtype predominates in the anterior pituitary. Using a similar technical approach, the aim of the present study was to establish the precise anatomical localization of cells synthetising the AT1A receptor in the adult rat brain. High AT1A messenger RNA expression was found in the vascular organ of the lamina terminalis, the median preoptic nucleus, the subfornical organ, the hypothalamic periventricular nucleus, the parvocellular parts of the paraventricular nucleus, the nucleus of the solitary tract and the area postrema, in agreement with previous autoradiographic studies, describing a high density of AT1 binding sites in these nuclei. In addition, AT1A messenger RNA expression was detected in several brain areas, where no AT1 binding was reported previously. Thus, we identify strong expression of AT1A messenger RNA expression in scattered cells of the lateral parts of the preoptic region, the lateral hypothalamus and several brainstem nuclei. In none of these structures was the AT1B messenger RNA detectable at the microscopic level. In conclusion, it is suggested that angiotensins may exert their central effects on body fluid and cardiovascular homeostasis mainly via the AT1A receptor subtype. PMID:9483539

  8. Rhythmic expression of functional MT1 melatonin receptors in the rat adrenal gland.

    PubMed

    Richter, Hans G; Torres-Farfan, Claudia; Garcia-Sesnich, Jocelyn; Abarzua-Catalan, Lorena; Henriquez, Mauricio G; Alvarez-Felmer, Mauro; Gaete, Fernando; Rehren, German E; Seron-Ferre, Maria

    2008-03-01

    We previously demonstrated that melatonin is involved in the regulation of adrenal glucocorticoid production in diurnal primates through activation of MT1 membrane-bound melatonin receptors. However, whether melatonin has a similar role in nocturnal rodents remains unclear. Using an integrative approach, here we show that the adult rat adrenal gland expresses a functional MT1 melatonin receptor in a rhythmic fashion. We found that: 1) expression of the cognate mRNA encoding for the MT1 membrane-bound melatonin receptor, displaying higher levels in the day/night transition (1800-2200 h); 2) expression of the predicted 37-kDa MT1 polypeptide in immunoblots from adrenals collected at 2200 h but not 1000 h; 3) no expression of the MT2 melatonin receptor mRNA and protein; 4) specific high-affinity 2-[(125)I]iodomelatonin binding in membrane fractions and frozen sections from adrenals collected at 2200 h but not 0800 h (dissociation constant = 14.22 +/- 1.23 pm; maximal binding capacity = 0.88 +/- 0.02 fmol/mg protein); and 5) in vitro clock time-dependent inhibition of ACTH-stimulated corticosterone production by 1-100 nm melatonin, which was reversed by 1 microm luzindole (a melatonin membrane receptor antagonist). Our findings indicate not only expression but also high amplitude diurnal variation of functional MT1 melatonin receptors in the rat adrenal gland. It is conceivable that plasma melatonin may play a role to fine-tune corticosterone production in nocturnal rodents, probably contributing to the down slope of the corticosterone rhythm. PMID:18039783

  9. Comparison of synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors and their gene expression in response to feeding in Ixodes scapularis (Ixodidae) vs. Ornithodoros turicata (Argasidae).

    PubMed

    Egekwu, N; Sonenshine, D E; Garman, H; Barshis, D J; Cox, N; Bissinger, B W; Zhu, J; M Roe, R

    2016-02-01

    Illumina GAII high-throughput sequencing was used to compare expressed genes for female synganglion neuropeptides, neuropeptide receptors and neurotransmitter receptors of the soft tick Ornithodoros turicata with the hard tick Ixodes scapularis. Gene ontology molecular level three mapping revealed no significant differences amongst the same categories represented in O. turicata and I. scapularis. Transcripts predicting 22 neuropeptides or their receptors in the O. turicata synganglion were similar to annotations for 23 neuropeptides or receptors previously identified from I scapularis, with minor exceptions. A transcript predicting ecdysis triggering hormone receptor was identified in O. turicata; transcripts encoding for proprotein convertase and glycoprotein B were identified in both species. Transcripts predicting the same neurotransmitter receptors were found in the synganglion of both species. Gene expression of the transcripts showed numerous differences in response to feeding. Major differences were observed in expression of genes believed important in regulating slow vs. rapid feeding, blood water elimination, cuticle synthesis plasticity and in signalling reproductive activity. Although the glutamate receptor was strongly upregulated in both species, the gamma aminobutyric acid receptor, which inhibits glutamate, was upregulated significantly only in I. scapularis. These differences are consistent with the slow vs. rapid action of the pharyngeal pump in the two species. PMID:26783017

  10. TRAIL Death Receptor-4 Expression Positively Correlates With the Tumor Grade in Breast Cancer Patients With Invasive Ductal Carcinoma

    SciTech Connect

    Sanlioglu, Ahter D.; Korcum, Aylin F.; Pestereli, Elif; Erdogan, Gulgun; Karaveli, Seyda; Savas, Burhan; Griffith, Thomas S.; Sanlioglu, Salih V.

    2007-11-01

    Purpose: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. Methods and Materials: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. Results: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. Conclusions: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.

  11. Chemokine receptor expression by inflammatory T cells in EAE

    PubMed Central

    Mony, Jyothi Thyagabhavan; Khorooshi, Reza; Owens, Trevor

    2014-01-01

    Chemokines direct cellular infiltration to tissues, and their receptors and signaling pathways represent targets for therapy in diseases such as multiple sclerosis (MS). The chemokine CCL20 is expressed in choroid plexus, a site of entry of T cells to the central nervous system (CNS). The CCL20 receptor CCR6 has been reported to be selectively expressed by CD4+ T cells that produce the cytokine IL-17 (Th17 cells). Th17 cells and interferon-gamma (IFNγ)-producing Th1 cells are implicated in induction of MS and its animal model experimental autoimmune encephalomyelitis (EAE). We have assessed whether CCR6 identifies specific inflammatory T cell subsets in EAE. Our approach was to induce EAE, and then examine chemokine receptor expression by cytokine-producing T cells sorted from CNS at peak disease. About 7% of CNS-infiltrating CD4+ T cells produced IFNγ in flow cytometric cytokine assays, whereas less than 1% produced IL-17. About 1% of CD4+ T cells produced both cytokines. CCR6 was expressed by Th1, Th1+17 and by Th17 cells, but not by CD8+ T cells. CD8+ T cells expressed CXCR3, which was also expressed by CD4+ T cells, with no correlation to cytokine profile. Messenger RNA for IFNγ, IL-17A, and the Th1 and Th17-associated transcription factors T-bet and RORγt was detected in both CCR6+ and CXCR3+ CD4+ T cells. IFNγ, but not IL-17A mRNA expression was detected in CD8+ T cells in CNS. CCR6 and CD4 were co-localized in spinal cord infiltrates by double immunofluorescence. Consistent with flow cytometry data some but not all CD4+ T cells expressed CCR6 within infiltrates. CD4-negative CCR6+ cells included macrophage/microglial cells. Thus we have for the first time directly studied CD4+ and CD8+ T cells in the CNS of mice with peak EAE, and determined IFNγ and IL17 expression by cells expressing CCR6 and CXCR3. We show that neither CCR6 or CXCR3 align with CD4 T cell subsets, and Th1 or mixed Th1+17 predominate in EAE. PMID:25071447

  12. Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

    PubMed

    Fan, Jifeng; Li, Jie; Fan, Qinbo

    2015-09-01

    Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation. PMID:26126997

  13. Fighting experience alters brain androgen receptor expression dependent on testosterone status

    PubMed Central

    Li, Cheng-Yu; Earley, Ryan L.; Huang, Shu-Ping; Hsu, Yuying

    2014-01-01

    Contest decisions are influenced by the outcomes of recent fights (winner–loser effects). Steroid hormones and serotonin are closely associated with aggression and therefore probably also play important roles in mediating winner–loser effects. In mangrove rivulus fish, Kryptolebias marmoratus, individuals with higher testosterone (T), 11-ketotestosterone and cortisol levels are more capable of winning, but titres of these hormones do not directly mediate winner–loser effects. In this study, we investigated the effects of winning/losing experiences on brain expression levels of the receptor genes for androgen (AR), oestrogen α/β (ERα/β), glucocorticoid (GR) and serotonin (5-HT1AR). The effect of contest experience on AR gene expression depended on T levels: repeated losses decreased, whereas repeated wins increased AR gene expression in individuals with low T but not in individuals with medium or high T levels. These results lend strong support for AR being involved in mediating winner–loser effects, which, in previous studies, were more detectable in individuals with lower T. Furthermore, the expression levels of ERα/β, 5-HT1AR and GR genes were higher in individuals that initiated contests against larger opponents than in those that did not. Overall, contest experience, underlying endocrine state and hormone and serotonin receptor expression patterns interacted to modulate contest decisions jointly. PMID:25320171

  14. Spatiotemporal expression of Nogo-66 receptor after focal cerebral ischemia

    PubMed Central

    Cao, Yue; Dong, Ya-xian; Xu, Jie; Chu, Guo-liang; Yang, Zhi-hua; Liu, Yan-ming

    2016-01-01

    NgR, the receptor for the neurite outgrowth inhibitor Nogo-66, plays a critical role in the plasticity and regeneration of the nervous system after injury such as ischemic stroke. In the present study, we used immunohistochemistry to investigate the regional expression of NgR in rat brain following middle cerebral artery occlusion (MCAO). NgR protein expression was not observed in the center of the lesion, but was elevated in the marginal zone compared with control and sham-operated rats. The cerebral cortex and hippocampus (CA1, CA2, and CA3) showed the greatest expression of NgR. Furthermore, NgR expression was higher in the ipsilesional hemisphere than on the control side in the same coronal section. Although time-dependent changes in NgR expression across brain regions had their own characteristics, the overall trend complied with the following rules: NgR expression changes with time showed two peaks and one trough; the first peak in expression appeared between 1 and 3 days after MCAO; expression declined at 5 days; and the second peak occurred at 28 days. PMID:26981102

  15. Regulation of luteinizing hormone receptor mRNA expression by a specific RNA binding protein in the ovary*

    PubMed Central

    Menon, K.M.J.; Nair, Anil K.; Wang, Lei; Peegel, Helle

    2009-01-01

    Summary The expression of LH receptor mRNA shows significant changes during different physiological states of the ovary. Previous studies from our laboratory have identified a post-transcriptional mechanism by which LH receptor mRNA is regulated following preovulatory LH surge or in response to hCG administration. A specific binding protein, identified as mevalonate kinase, binds to the open reading frame of LH receptor mRNA. The protein binding site is localized to nucleotides 203–220 of the LH receptor mRNA and exhibits a high degree of specificity. The expression levels of the protein show an inverse relationship to the LH receptor mRNA levels. The hCG-induced down-regulation of LH receptor mRNA can be mimicked by increasing the intracellular levels of cyclic AMP by a phosphodiesterase inhibitor. An in vitro mRNA decay assay showed that addition of the binding protein to the decay system caused accelerated LH receptor mRNA decay. Our results therefore show that LH receptor mRNA expression in the ovary is regulated post-transcriptionally by altering the rate of mRNA degradation by a specific mRNA binding protein. PMID:17055149

  16. Prolactinoma ErbB receptor expression and targeted therapy for aggressive tumors.

    PubMed

    Cooper, Odelia; Mamelak, Adam; Bannykh, Serguei; Carmichael, John; Bonert, Vivien; Lim, Stephen; Cook-Wiens, Galen; Ben-Shlomo, Anat

    2014-06-01

    As ErbB signaling is a determinant of prolactin synthesis, role of ErbB receptors was tested for prolactinoma outcomes and therapy. The objective of this study was to characterize ErbB receptor expression in prolactinomas and then perform a pilot study treating resistant prolactinomas with a targeted tyrosine kinase inhibitor (TKI). Retrospective analysis of prolactinomas and pilot study for dopamine agonist resistant prolactinomas in tertiary referral center. We performed immunofluorescent staining of a tissue array of 29 resected prolactinoma tissues for EGFR, ErbB2, ErbB3, and ErbB4 correlated with clinical features. Two patients with aggressive resistant prolactinomas enrolled and completed trial. They received lapatinib 1,250 mg daily for 6 months with tumor and hormone assessments. Main outcome measures were positive tumor staining of respective ErbB receptors, therapeutic reduction of prolactin levels and tumor shrinkage. Treated PRL levels and tumor volumes were suppressed in both subjects treated with TKI. EGFR expression was positive in 82 % of adenomas, ErbB2 in 92 %, ErbB3 in 25 %, and ErbB4 in 71 %, with ErbB2 score > EGFR > ErbB4 > ErbB3. Higher ErbB3 expression was associated with optic chiasm compression (p = 0.03), suprasellar extension (p = 0.04), and carotid artery encasement (p = 0.01). Higher DA response rates were observed in tumors with higher ErbB3 expression. Prolactinoma expression of specific ErbB receptors is associated with tumor invasion, symptoms, and response to dopamine agonists. Targeting ErbB receptors may be effective therapy in patients with resistant prolactinomas. PMID:24287797

  17. Beta-Adrenergic Receptor Expression in Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, Ronald B.; Bridge, K.; Vaughn, J. R.

    1999-01-01

    beta-adrenergic receptor (bAR) agonists presumably exert their physiological action on skeletal muscle cells through the bAR. Since the signal generated by the bAR is cyclic AMP (cAMP), experiments were initiated in primary chicken muscle cell cultures to determine if artificial elevation of intracellular cAMP by treatment with forskolin would alter the population of bAR expressed on the surface of muscle cells. Chicken skeletal muscle cells after 7 days in culture were employed for the experiments because muscle cells have attained a steady state with respect to muscle protein metabolism at this stage. Cells were treated with 0-10 uM forskolin for a total of three days. At the end of the 1, 2, and 3 day treatment intervals, the concentration of cAMP and the bAR population were measured. Receptor population was measured in intact muscle cell cultures as the difference between total binding of [H-3]CGP-12177 and non-specific binding of [H-3]CGP-12177 in the presence of 1 uM propranolol. Intracellular cAMP concentration was measured by radioimmunoassay. The concentration of cAMP in forskolin-treated cells increased up to 10-fold in a dose dependent manner. Increasing concentrations of forskolin also led to an increase in (beta)AR population, with a maximum increase of approximately 50% at 10 uM. This increase in (beta)AR population was apparent after only 1 day of treatment, and the pattern of increase was maintained for all 3 days of the treatment period. Thus, increasing the intracellular concentration of cAMP leads to up-regulation of (beta)AR population. Clenbuterol and isoproterenol gave similar effects on bAR population. The effect of forskolin on the quantity and apparent synthesis rate of the heavy chain of myosin (mhc) were also investigated. A maximum increase of 50% in the quantity of mhc was observed at 0.2 UM forskolin, but higher concentrations of forskolin reduced the quantity of mhc back to control levels.

  18. The p75 neurotrophin receptor localization in blood-CSF barrier: expression in choroid plexus epithelium

    PubMed Central

    2011-01-01

    Background The presence of neurotrophins and their receptors Trk family has been reported in the choroid plexus. High levels of Nerve Growth Factor (NGF), Neurotrophin-4 (NT-4) and TrkB receptor were detected, while nothing was know about p75 neurotrophin receptor (p75NTR) in the choroid plexus epithelial cells. In neurons, p75NTR receptor has a dual function: promoting survival together with TrkA in response to NGF, and inducing apoptotic signaling through p75NTR. We postulated that p75NTR may also affect the survival pathways in the choroid plexus and also undergoes regulated proteolysis with metalloproteases. Results Here, we demonstrated the presence of p75NTR receptor in the choroid plexus epithelial cells. The p75NTR receptor would be involved in cell death mechanisms and in the damaged induced by amyloid beta (Aβ) in the choroid plexus and finally, we propose an essential role of p75NTR in the Aβ transcytosis through out choroid plexus barrier. Conclusions The presence analysis reveals the new localization of p75NTR in the choroid plexus and, the distribution mainly in the cytoplasm and cerebrospinal fluid (CSF) side of the epithelial cells. We propose that p75NTR receptor plays a role in the survival pathways and Aβ-induced cell death. These data suggest that p75NTR dysfunction play an important role in the pathogenesis of brain diseases. The importance and novelty of this expression expands a new role of p75NTR. PMID:21569322

  19. Efficient silkworm expression of human GPCR (nociceptin receptor) by a Bombyx mori bacmid DNA system

    SciTech Connect

    Kajikawa, Mizuho; Sasaki, Kaori; Wakimoto, Yoshitaro; Toyooka, Masaru; Motohashi, Tomoko; Shimojima, Tsukasa; Takeda, Shigeki; Park, Enoch Y.; Maenaka, Katsumi

    2009-07-31

    Guanine nucleotide-binding protein (G protein) coupled receptors (GPCRs) are frequently expressed by a baculovirus expression vector system (BEVS). We recently established a novel BEVS using the bacmid system of Bombyx mori nucleopolyhedrovirus (BmNPV), which is directly applicable for protein expression in silkworms. Here, we report the first example of GPCR expression in silkworms by the simple injection of BmNPV bacmid DNA. Human nociceptin receptor, an inhibitory GPCR, and its fusion protein with inhibitory G protein alpha subunit (G{sub i}{alpha}) were both successfully expressed in the fat bodies of silkworm larvae as well as in the BmNPV viral fraction. Its yield was much higher than that from Sf9 cells. The microsomal fractions including the nociceptin receptor fusion, which are easily prepared by only centrifugation steps, exhibited [{sup 35}S]GTP{gamma}S-binding activity upon specific stimulation by nociceptin. Therefore, this rapid method is easy-to-use and has a high expression level, and thus will be an important tool for human GPCR production.

  20. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    PubMed Central

    Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

    2012-01-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  1. Effects of daidzein on messenger ribonucleic Acid expression of gonadotropin receptors in chicken ovarian follicles.

    PubMed

    Liu, H Y; Zhang, C Q

    2008-03-01

    Effects of daidzein on expression of mRNA of gonadotropin receptors [follicle-stimulating hormone receptor (FSHR), luteinizing hormone receptor (LHR)] were evaluated in ovarian follicles of ISA laying hens that were 13 mo old in the postpeak period of egg laying. The hens were randomly allocated as control and daidzein-treated groups, with daidzein supplemented to the basal diet at the level of 10 mg/kg for 7 wk. The granulosa layers of preovulatory follicles (F1, F2, F3, F4, F5) and follicular layers of the small yellow follicle (SYF), large white follicle (LWF), and atretic follicle were collected. The mRNA expression of related genes was measured by semiquantitative reverse transcription PCR. Results showed that daidzein significantly increased the egg-laying rate (P < 0.05) and the number of SYF and LWF (P < 0.05). The relative abundance of the FSHR mRNA decreased in the granulosa layers from F5 to F1, but LHR mRNA displayed the opposite trend in developmental changes. Treatment with daidzein resulted in increased expression of FSHR mRNA in LWF, SYF, and granulosa layers of F4 to F2 and LHR mRNA in granulosa layers of F4 and F1 (P < 0.05). These results indicated that dietary supplementation of daidzein upregulated mRNA expression of gonadotropin receptors to improve follicle development in chicken developing follicles and laying performance after the peak laying period. PMID:18281582

  2. Progesterone receptor expression declines in the guinea pig uterus during functional progesterone withdrawal and in response to prostaglandins.

    PubMed

    Welsh, Toni N; Hirst, Jonathan J; Palliser, Hannah; Zakar, Tamas

    2014-01-01

    Progesterone withdrawal is essential for parturition, but the mechanism of this pivotal hormonal change is unclear in women and other mammals that give birth without a pre-labor drop in maternal progesterone levels. One possibility suggested by uterine tissue analyses and cell culture models is that progesterone receptor levels change at term decreasing the progesterone responsiveness of the myometrium, which causes progesterone withdrawal at the functional level and results in estrogen dominance enhancing uterine contractility. In this investigation we have explored whether receptor mediated functional progesterone withdrawal occurs during late pregnancy and labor in vivo. We have also determined whether prostaglandins that induce labor cause functional progesterone withdrawal by altering myometrial progesterone receptor expression. Pregnant guinea pigs were used, since this animal loses progesterone responsiveness at term and gives birth in the presence of high maternal progesterone level similarly to primates. We found that progesterone receptor mRNA and protein A and B expression decreased in the guinea pig uterus during the last third of gestation and in labor. Prostaglandin administration reduced while prostaglandin synthesis inhibitor treatment increased progesterone receptor A protein abundance. Estrogen receptor-1 protein levels remained unchanged during late gestation, in labor and after prostaglandin or prostaglandin synthesis inhibitor administration. Steroid receptor levels were higher in the non-pregnant than in the pregnant uterine horns. We conclude that the decreasing expression of both progesterone receptors A and B is a physiological mechanism of functional progesterone withdrawal in the guinea pig during late pregnancy and in labor. Further, prostaglandins administered exogenously or produced endogenously stimulate labor in part by suppressing uterine progesterone receptor A expression, which may cause functional progesterone withdrawal, promote

  3. Regulation of retinoid X receptor gamma expression by fed state in mouse liver.

    PubMed

    Park, Sangkyu; Lee, Yoo Jeong; Ko, Eun Hee; Kim, Jae-Woo

    2015-02-27

    Glucose metabolism is balanced by glycolysis and gluconeogenesis with precise control in the liver. The expression of genes related to glucose metabolism is regulated primarily by glucose and insulin at transcriptional level. Nuclear receptors play important roles in regulating the gene expression of glucose metabolism at transcriptional level. Some of these nuclear receptors form heterodimers with RXRs to bind to their specific regulatory elements on the target promoters. To date, three isotypes of RXRs have been identified; RXRα, RXRβ and RXRγ. However, their involvement in the interactions with other nuclear receptors in the liver remains unclear. In this study, we found RXRγ is rapidly induced after feeding in the mouse liver, indicating a potential role of RXRγ in controlling glucose or lipid metabolism in the fasting-feeding cycle. In addition, RXRγ expression was upregulated by glucose in primary hepatocytes. This implies that glucose metabolism governed by RXRγ in conjunction with other nuclear receptors. The luciferase reporter assay showed that RXRγ as well as RXRα increased SREBP-1c promoter activity in hepatocytes. These results suggest that RXRγ may play an important role in tight control of glucose metabolism in the fasting-feeding cycle. PMID:25637539

  4. Altered sensitivity to excitotoxic cell death and glutamate receptor expression between two commonly studied mouse strains

    PubMed Central

    Finn, Rozzy; Kovács, Attila D.; Pearce, David A.

    2011-01-01

    Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders including ischemia, epilepsy, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/SvEv mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to NMDA-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent upon the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity are likely due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/SvEv mouse backgrounds. PMID:20544821

  5. Clinical Relevance of VPAC1 Receptor Expression in Early Arthritis: Association with IL-6 and Disease Activity

    PubMed Central

    Seoane, Iria V.; Ortiz, Ana M.; Piris, Lorena; Lamana, Amalia; Juarranz, Yasmina; García-Vicuña, Rosario; González-Álvaro, Isidoro; Gomariz, Rosa P.; Martínez, Carmen

    2016-01-01

    Background The vasoactive intestinal peptide (VIP) receptors VPAC1 and VPAC2 mediate anti-inflammatory and immunoregulatory responses in rheumatoid arthritis (RA). Data on the expression of these receptors could complement clinical assessment in the management of RA. Our goal was to investigate the correlation between expression of both receptors and the 28-Joint Disease Activity Score (DAS28) in peripheral blood mononuclear cells (PBMCs) from patients with early arthritis (EA). We also measured expression of IL-6 to evaluate the association between VIP receptors and systemic inflammation. Methods We analyzed 250 blood samples collected at any of the 5 scheduled follow-up visits from 125 patients enrolled in the Princesa Early Arthritis Register Longitudinal study. Samples from 22 healthy donors were also analyzed. Sociodemographic, clinical, and therapeutic data were systematically recorded. mRNA expression levels were determined using real-time PCR. Then, longitudinal multivariate analyses were performed. Results PBMCs from EA patients showed significantly higher expression of VPAC2 receptors at baseline compared to healthy donors (p<0.001). With time, however, VPAC2 expression tended to be significantly lower while VPAC1 receptor expression increased in correlation with a reduction in DAS28 index. Our results reveal that more severe inflammation, based on high levels of IL-6, is associated with lower expression of VPAC1 (p<0.001) and conversely with increased expression of VPAC2 (p<0.001). A major finding of this study is that expression of VPAC1 is lower in patients with increased disease activity (p = 0.001), thus making it possible to differentiate between patients with various degrees of clinical disease activity. Conclusion Patients with more severe inflammation and higher disease activity show lower levels of VPAC1 expression, which is associated with patient-reported impairment. Therefore, VPAC1 is a biological marker in EA. PMID:26881970

  6. Distinct expression profiles of three melatonin receptors during early development and metamorphosis in the flatfish Solea senegalensis.

    PubMed

    Lan-Chow-Wing, Olivier; Confente, Francesca; Herrera-Pérez, Patricia; Isorna, Esther; Chereguini, Olvido; Rendón, Maria del Carmen; Falcón, Jack; Muñoz-Cueto, José A

    2014-01-01

    Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2-6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis. PMID:25402642

  7. Distinct Expression Profiles of Three Melatonin Receptors during Early Development and Metamorphosis in the Flatfish Solea senegalensis

    PubMed Central

    Lan-Chow-Wing, Olivier; Confente, Francesca; Herrera-Pérez, Patricia; Isorna, Esther; Chereguini, Olvido; Rendón, Maria del Carmen; Falcón, Jack; Muñoz-Cueto, José A.

    2014-01-01

    Melatonin actions are mediated through G protein-coupled transmembrane receptors. Recently, mt1, mt2, and mel1c melatonin receptors were cloned in the Senegalese sole. Here, their day-night and developmental expressions were analyzed by quantitative PCR. These results revealed distinct expression patterns of each receptor through development. mel1c transcripts were more abundant in unfertilized ovulated oocytes and declined during embryonic development. mt1 and mt2 expression was higher at the earliest stages (2–6 days post-fertilization), decreasing before (mt2) or during (mt1) metamorphosis. Only mt1 and mel1c expression exhibited day-night variations, with higher nocturnal mRNA levels. These results suggest different roles and transcriptional regulation of these melatonin receptors during flatfish development and metamorphosis. PMID:25402642

  8. Hepatic Aryl Hydrocarbon Receptor Attenuates Fibroblast Growth Factor 21 Expression.

    PubMed

    Girer, Nathaniel G; Murray, Iain A; Omiecinski, Curtis J; Perdew, Gary H

    2016-07-15

    The Aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor involved in many physiological processes. Several studies indicate that AHR is also involved in energy homeostasis. Fibroblast growth factor 21 (FGF21) is an important regulator of the fasting and feeding responses. When administered to various genetic and diet-induced mouse models of obesity, FGF21 can attenuate obesity-associated morbidities. Here, we explore the role of AHR in hepatic Fgf21 expression through the use of a conditional, hepatocyte-targeted AHR knock-out mouse model (Cre(Alb)Ahr(Fx/Fx)). Compared with the congenic parental strain (Ahr(Fx/Fx)), non-fasted Cre(Alb)Ahr(Fx/Fx) mice exhibit a 4-fold increase in hepatic Fgf21 expression, as well as elevated expression of the FGF21-target gene Igfbp1 Furthermore, in vivo agonist activation of AHR reduces hepatic Fgf21 expression during a fast. The Fgf21 promoter contains several putative dioxin response elements (DREs). Using EMSA, we demonstrate that the AHR-ARNT heterodimer binds to a specific DRE that overlaps binding sequences for peroxisome proliferator-activated receptor α (PPARα), carbohydrate response element-binding protein (ChREBP), and cAMP response element-binding protein, hepatocyte specific (CREBH). In addition, we reveal that agonist-activated AHR impairs PPARα-, ChREBP-, and CREBH-mediated promoter activity in Hepa-1 cells. Accordingly, agonist treatment in Hepa-1 cells ablates potent ER stress-driven Fgf21 expression, and pre-treatment with AHR antagonist blocks this effect. Finally, we show that pre-treatment of primary human hepatocytes with AHR agonist diminishes PPARα-, glucose-, and ER stress-driven induction of FGF21 expression, indicating the effect is not mouse-specific. Together, our data show that AHR contributes to hepatic energy homeostasis, partly through the regulation of FGF21 expression and signaling. PMID:27226639

  9. Hypoxia induces PDK4 gene expression through induction of the orphan nuclear receptor ERRγ.

    PubMed

    Lee, Ja Hee; Kim, Eun-Jin; Kim, Don-Kyu; Lee, Ji-Min; Park, Seung Bum; Lee, In-Kyu; Harris, Robert A; Lee, Mi-Ock; Choi, Hueng-Sik

    2012-01-01

    Multiple cellular signaling pathways that control metabolism and survival are activated when cell are incubated under hypoxic conditions. Activation of the hypoxia inducible factor (HIF)-1 promotes expression of genes that increase the capacity to cope with the stress imposed by a reduced oxygen environment. Here we show that the orphan nuclear receptor estrogen related receptor γ (ERRγ) plays a critical role in hypoxia-mediated activation of pyruvate dehydrogenase kinase 4 (PDK4) gene expression. ERRγ mRNA and protein levels were increased by hypoxia or desferrioxamine (DFO) treatment in hepatoma cell lines. Co-expression of HIF-1α and β increased ERRγ promoter activity as well as mRNA expression, while knockdown of endogenous HIF-1α reduced the hypoxia-mediated induction of ERRγ. In addition, hypoxia also increased the promoter activity and mRNA level of PDK4 in HepG2 cells. Adenovirus mediated-overexpression of ERRγ specifically increased PDK4 gene expression, while ablation of endogenous ERRγ significantly decreased hypoxia-mediated induction of PDK4 gene expression. Finally, GSK5182, an inverse agonist of ERRγ, strongly inhibited the hypoxia-mediated induction of PDK4 protein and promoter activity. Regulation of the transcriptional activity of ERRγ may provide a therapeutic approach for the regulation of PDK4 gene expression under hypoxia. PMID:23050013

  10. Hypoxia Induces PDK4 Gene Expression through Induction of the Orphan Nuclear Receptor ERRγ

    PubMed Central

    Lee, Ji-Min; Park, Seung Bum; Lee, In-Kyu; Harris, Robert A.; Lee, Mi-Ock; Choi, Hueng-Sik

    2012-01-01

    Multiple cellular signaling pathways that control metabolism and survival are activated when cell are incubated under hypoxic conditions. Activation of the hypoxia inducible factor (HIF)-1 promotes expression of genes that increase the capacity to cope with the stress imposed by a reduced oxygen environment. Here we show that the orphan nuclear receptor estrogen related receptor γ (ERRγ) plays a critical role in hypoxia–mediated activation of pyruvate dehydrogenase kinase 4 (PDK4) gene expression. ERRγ mRNA and protein levels were increased by hypoxia or desferrioxamine (DFO) treatment in hepatoma cell lines. Co-expression of HIF-1α and β increased ERRγ promoter activity as well as mRNA expression, while knockdown of endogenous HIF-1α reduced the hypoxia-mediated induction of ERRγ. In addition, hypoxia also increased the promoter activity and mRNA level of PDK4 in HepG2 cells. Adenovirus mediated-overexpression of ERRγ specifically increased PDK4 gene expression, while ablation of endogenous ERRγ significantly decreased hypoxia-mediated induction of PDK4 gene expression. Finally, GSK5182, an inverse agonist of ERRγ, strongly inhibited the hypoxia-mediated induction of PDK4 protein and promoter activity. Regulation of the transcriptional activity of ERRγ may provide a therapeutic approach for the regulation of PDK4 gene expression under hypoxia. PMID:23050013

  11. Lamina-specific alterations in cortical GABA(A) receptor subunit expression in schizophrenia.

    PubMed

    Beneyto, Monica; Abbott, Andrew; Hashimoto, Takanori; Lewis, David A

    2011-05-01

    Dysfunction of the dorsolateral prefrontal cortex (DLPFC) in schizophrenia is associated with lamina-specific alterations in particular subpopulations of interneurons. In pyramidal cells, postsynaptic γ-aminobutyric acid (GABA(A)) receptors containing different α subunits are inserted preferentially in distinct subcellular locations targeted by inputs from specific interneuron subpopulations. We used in situ hybridization to quantify the laminar expression of α1, α2, α3, and α5 subunit, and of β1-3 subunit, mRNAs in the DLFPC of schizophrenia, and matched normal comparison subjects. In subjects with schizophrenia, mean GABA(A) α1 mRNA expression was 17% lower in layers 3 and 4, α2 expression was 14% higher in layer 2, α5 expression was 15% lower in layer 4, and α3 expression did not differ relative to comparison subjects. The mRNA expression of β2, which preferentially assembles with α1 subunits, was also 20% lower in layers 3 and 4, whereas β1 and β3 mRNA levels were not altered in schizophrenia. These expression differences were not attributable to medication effects or other potential confounds. These findings suggest that GABA neurotransmission in the DLPFC is altered at the postsynaptic level in a receptor subunit- and layer-specific manner in subjects with schizophrenia and support the hypothesis that GABA neurotransmission in this illness is predominantly impaired in certain cortical microcircuits. PMID:20843900

  12. Expression of lactoferrin receptors is increased in the mesencephalon of patients with Parkinson disease.

    PubMed Central

    Faucheux, B A; Nillesse, N; Damier, P; Spik, G; Mouatt-Prigent, A; Pierce, A; Leveugle, B; Kubis, N; Hauw, J J; Agid, Y

    1995-01-01

    The degeneration of nigral dopaminergic neurons in Parkinson disease is believed to be associated with oxidative stress. Since iron levels are increased in the substantia nigra of parkinsonian patients and this metal catalyzes the formation of free radicals, it may be involved in the mechanisms of nerve cell death. The cause of nigral iron increase is not understood. Iron acquisition by neurons may occur from iron-transferrin complexes with a direct interaction with specific membrane receptors, but recent results have shown a low density of transferrin receptors in the substantia nigra. To investigate whether neuronal death in Parkinson disease may be associated with changes in a pathway supplementary to that of transferrin, lactoferrin (lactotransferrin) receptor expression was studied in the mesencephalon. In this report we present evidence from immunohistochemical staining of postmortem human brain tissue that lactoferrin receptors are localized on neurons (perikarya, dendrites, axons), cerebral microvasculature, and, in some cases, glial cells. In parkinsonian patients, lactoferrin receptor immunoreactivity on neurons and microvessels was increased and more pronounced in those regions of the mesencephalon where the loss of dopaminergic neurons is severe. Moreover, in the substantia nigra, the intensity of immunoreactivity on neurons and microvessels was higher for patients with higher nigral dopaminergic loss. These data suggest that lactoferrin receptors on vulnerable neurons may increase intraneuronal iron levels and contribute to the degeneration of nigral dopaminergic neurons in Parkinson disease. Images Fig. 1 Fig. 2 PMID:7568181

  13. Association of estrogen receptor-α and progesterone receptor A expression with hormonal mammary carcinogenesis: role of the host microenvironment

    PubMed Central

    Montero Girard, Guadalupe; Vanzulli, Silvia I; Cerliani, Juan Pablo; Bottino, María Cecilia; Bolado, Julieta; Vela, Jorge; Becu-Villalobos, Damasia; Benavides, Fernando; Gutkind, Silvio; Patel, Vyomesh; Molinolo, Alfredo; Lanari, Claudia

    2007-01-01

    Introduction Medroxyprogesterone acetate (MPA) induces estrogen receptor (ER)-positive and progesterone receptor (PR)-positive ductal invasive mammary carcinomas in BALB/c mice. We sought to reproduce this MPA cancer model in C57BL/6 mice because of their widespread use in genetic engineering. Within this experimental setting, we studied the carcinogenic effects of MPA, the morphologic changes in mammary glands that are induced by MPA and progesterone, and the levels of ER and PR expression in MPA-treated and progesterone-treated mammary glands. Finally, we evaluated whether the differences found between BALB/c and C57BL/6 mouse strains were due to intrinsic differences in epithelial cells. Methods The carcinogenic effect of MPA was evaluated in C57BL/6 mice using protocols proven to be carcinogenic in BALB/c mice. In addition, BALB/c and C57BL/6 females were treated with progesterone or MPA for 1 or 2 months, and mammary glands were excised for histologic studies and for immunohistochemical and Western blot evaluation of ER and PR. Hormone levels were determined by radioimmunoassay. Isolated mammary epithelial cells were transplanted into cleared fat pads of 21-day-old female Swiss nu/nu mice or control congenic animals. Results MPA failed to induce mammary carcinomas or significant morphologic changes in the mammary glands of C57BL/6 mice. The expression of ER-α and PR isoform A in virgin mice was surprisingly much higher in BALB/c than in C57BL/6 mammary glands, and both receptors were downregulated in progestin-treated BALB/c mice (P < 0.05). PR isoform B levels were low in virgin control mice and increased after progestin treatment in both strains. ER-β expression followed a similar trend. No differences in hormone levels were found between strains. Surprisingly, the transplantation of the epithelial mammary gland cells of both strains into the cleared fat pads of Swiss (nu/nu) mice abolished the mammary gland morphologic differences and the ER and PR

  14. Up regulation of AT4 receptor levels in carotid arteries following balloon injury.

    PubMed

    Moeller, I; Clune, E F; Fennessy, P A; Bingley, J A; Albiston, A L; Mendelsohn, F A; Chai, S Y

    1999-08-31

    Angiotensin IV, (V-Y-I-H-P-F), binds to AT4 receptors in blood vessels to induce vasodilatation and proliferation of cultured bovine endothelial cells. This latter effect may be important not only in developing tissues but also in injured vessels undergoing remodelling. In the present study, using normal rabbit carotid arteries, we detected AT4 receptors in vascular smooth muscle cells and in the vasa vasorum of the adventitia. Very low receptor levels were observed in the endothelial cells. In keeping with the described binding specificity of AT4 receptors, unlabelled angiotensin IV competed for [125I]angiotensin IV binding in the arteries, with an IC50 of 1.4 nM, whereas angiotensin II and angiotensin III were weaker competitors. Within the first week following endothelial denudation of the carotid artery by balloon catheter, AT4 receptor binding in the media increased to approximately 150% of control tissue. AT4 receptor binding further increased in the media, large neointima and re-endothelialized cell layer to 223% at 20 weeks after injury. In view of the known trophic effects of angiotensin IV, the elevated expression of AT4 receptors, in both the neointima and media of arteries, following balloon injury to the endothelium, suggests a role for the peptide in the adaptive response and remodelling of the vascular wall following damage. PMID:10498341

  15. Expression of estrogen receptor subtypes in rat pituitary gland during pregnancy and lactation.

    PubMed

    Vaillant, Colette; Chesnel, Franck; Schausi, Diane; Tiffoche, Christophe; Thieulant, Marie-Lise

    2002-11-01

    The aim of this study was to examine whether the expression levels of mRNA of the three estrogen receptor (ER) subtypes, ERalpha, ERbeta, and truncated ER product-1 (TERP-1) found in the rat pituitary gland were modified during gestation, lactation, and postlactation periods. By using relative quantitative RT-PCR, we found that ERalpha mRNA significantly peaked in midpregnancy. However, the ERalpha protein level remained constant. ERbeta gene expression did not change throughout pregnancy, suggesting that it was not related to estradiol levels during this reproductive period. In contrast, both TERP-1 mRNA and protein levels dramatically increased throughout the second half of gestation, being faintly detectable in early pregnancy. TERP-1 expression was rapidly reversed by lactation, whereas neither pituitary ERalpha nor ERbeta relative levels were significantly altered. In addition, pup removal for 24-96 h on d 9 postpartum significantly reduced the expression of both ERalpha and ERbeta mRNA compared with that in lactating animals, but the expression of TERP-1 mRNA was no longer detected. Collectively, our data indicate that 1) TERP-1, ERalpha, and ERbeta expression levels are differentially regulated in the pituitary; 2) TERP-1 is variably expressed depending on the hormonal environment related to the estrous cycle, pregnancy, and lactation; and 3) TERP-1/ERalpha ratios dramatically change depending on reproductive periods, suggesting a critical role for TERP-1 in reproductive events. PMID:12399419

  16. Cyclic AMP-receptor protein activates aerobactin receptor IutA expression in Vibrio vulnificus.

    PubMed

    Kim, Choon-Mee; Kim, Seong-Jung; Shin, Sung-Heui

    2012-04-01

    The ferrophilic bacterium Vibrio vulnificus can utilize the siderophore aerobactin of Escherichia coli for iron acquisition via its specific receptor IutA. This siderophore piracy by V. vulnificus may contribute to its survival and proliferation, especially in mixed bacterial environments. In this study, we examined the effects of glucose, cyclic AMP (cAMP), and cAMP-receptor protein (Crp) on iutA expression in V. vulnificus. Glucose dose-dependently repressed iutA expression. A mutation in cya encoding adenylate cyclase required for cAMP synthesis severely repressed iutA expression, and this change was recovered by in trans complementing cya or the addition of exogenous cAMP. Furthermore, a mutation in crp encoding Crp severely repressed iutA expression, and this change was recovered by complementing crp. Accordingly, glucose deprivation under iron-limited conditions is an environmental signal for iutA expression, and Crp functions as an activator that regulates iutA expression in response to glucose availability. PMID:22538662

  17. Insulin decreases atherosclerosis by inducing endothelin receptor B expression

    PubMed Central

    Park, Kyoungmin; Mima, Akira; Li, Qian; Rask-Madsen, Christian; He, Pingnian; Mizutani, Koji; Katagiri, Sayaka; Maeda, Yasutaka; Wu, I-Hsien; Khamaisi, Mogher; Preil, Simone Rordam; Maddaloni, Ernesto; Sørensen, Ditte; Rasmussen, Lars Melholt; Huang, Paul L.; King, George L.

    2016-01-01

    Endothelial cell (EC) insulin resistance and dysfunction, caused by diabetes, accelerates atherosclerosis. It is unknown whether specifically enhancing EC-targeted insulin action can decrease atherosclerosis in diabetes. Accordingly, overexpressing insulin receptor substrate-1 (IRS1) in the endothelia of Apoe−/− mice (Irs1/Apoe−/−) increased insulin signaling and function in the aorta. Atherosclerosis was significantly reduced in Irs1/ApoE−/− mice on diet-induced hyperinsulinemia and hyperglycemia. The mechanism of insulin’s enhanced antiatherogenic actions in EC was related to remarkable induction of NO action, which increases endothelin receptor B (EDNRB) expression and intracellular [Ca2+]. Using the mice with knockin mutation of eNOS, which had Ser1176 mutated to alanine (AKI), deleting the only known mechanism for insulin to activate eNOS/NO pathway, we observed that IRS1 overexpression in the endothelia of Aki/ApoE−/− mice significantly decreased atherosclerosis. Interestingly, endothelial EDNRB expression was selectively reduced in intima of arteries from diabetic patients and rodents. However, endothelial EDNRB expression was upregulated by insulin via P13K/Akt pathway. Finally EDNRB deletion in EC of Ldlr−/− and Irs1/Ldlr−/− mice decreased NO production and accelerated atherosclerosis, compared with Ldlr−/− mice. Accelerated atherosclerosis in diabetes may be reduced by improving insulin signaling selectively via IRS1/Akt in the EC by inducing EDNRB expression and NO production. PMID:27200419

  18. Modeling the effects of HER/ErbB1-3 co-expression on receptor dimerization and biological response

    SciTech Connect

    Shankaran, Harish; Wiley, H. S.; Resat, Haluk

    2006-06-01

    The human epidermal growth factor receptor (HER/ErbB) system comprises the epidermal growth factor receptor (EGFR/HER1) and three other homologues viz. HER2-4. This receptor system plays a critical role in cell proliferation and differentiation and receptor over-expression can be associated with poor prognosis in cancers of the epithelium. Here, we examine the effect of co-expressing varying levels of HER1-3 on the receptor dimerization patterns using a detailed kinetic model for ErbB heterodimerization and trafficking. Our results indicate that co-expression of EGFR with HER2 or HER3 biases signaling to the cell surface and retards signal down-regulation. In addition, simultaneous co-expression of HER1-3 leads to preferential formation of HER2-HER3 heterodimers, which are known to be potent inducers of cell growth and transformation. Analysis of the parameter dependencies in the model reveals that measurements of HER3 phosphorylation and HER2 internalization ratio may prove to be especially useful for the estimation of critical model parameters. Further, we examined the effect of receptor dimerization patterns on cell phenotype using a simple phenomenological model. Results indicate that co-expression of EGFR with HER2 and HER3 at low to moderate levels may enable cells to match the phenotype of a high HER2 expresser.

  19. Expression of metabotropic glutamate receptor 4 in osteosarcoma

    PubMed Central

    WANG, SHUO; WEI, XING; CHEN, BINGYAO; ZHAO, MIN; SONG, GUANGZE; ZHANG, ZENGLIANG; LI, NAN

    2016-01-01

    Metabotropic glutamate receptor 4 (mGluR4) has been associated with the pathogenesis of osteosarcoma. The aim of this study was to investigate mGluR4 expression and its clinical significance in osteosarcoma patients. mGluR4 expression was investigated using immunohistochemistry (IHC) in 58 osteosarcomas and 32 giant-cell tumors of bone. The correlations between mGluR4 expression and clinicopathological characteristics were analyzed with the Chi-squared test and survival curves were generated using the Kaplan-Meier method. The IHC results demonstrated that 20.69% (12/58) of the osteosarcomas and 43.75% (14/32) of the giant-cell tumors were mGluR4-positive. The statistical analysis revealed that mGluR4 expression was correlated with gender, age, Enneking stage and tumor volume in osteosarcomas (P<0.05). In the multivariate stepwise Cox regression analysis, Enneking stage was found to be statistically significantly associated with survival (P<0.05) and the survival analysis demonstrated that the survival probability was significantly higher in patients with higher mGluR4 expression compared with those with lower expression (P<0.05). Therefore, mGluR4 expression may be used to estimate the prognosis of osteosarcoma patients. PMID:26870360

  20. Human neural progenitors express functional lysophospholipid receptors that regulate cell growth and morphology

    PubMed Central

    Hurst, Jillian H; Mumaw, Jennifer; Machacek, David W; Sturkie, Carla; Callihan, Phillip; Stice, Steve L; Hooks, Shelley B

    2008-01-01

    Background Lysophospholipids regulate the morphology and growth of neurons, neural cell lines, and neural progenitors. A stable human neural progenitor cell line is not currently available in which to study the role of lysophospholipids in human neural development. We recently established a stable, adherent human embryonic stem cell-derived neuroepithelial (hES-NEP) cell line which recapitulates morphological and phenotypic features of neural progenitor cells isolated from fetal tissue. The goal of this study was to determine if hES-NEP cells express functional lysophospholipid receptors, and if activation of these receptors mediates cellular responses critical for neural development. Results Our results demonstrate that Lysophosphatidic Acid (LPA) and Sphingosine-1-phosphate (S1P) receptors are functionally expressed in hES-NEP cells and are coupled to multiple cellular signaling pathways. We have shown that transcript levels for S1P1 receptor increased significantly in the transition from embryonic stem cell to hES-NEP. hES-NEP cells express LPA and S1P receptors coupled to Gi/o G-proteins that inhibit adenylyl cyclase and to Gq-like phospholipase C activity. LPA and S1P also induce p44/42 ERK MAP kinase phosphorylation in these cells and stimulate cell proliferation via Gi/o coupled receptors in an Epidermal Growth Factor Receptor (EGFR)- and ERK-dependent pathway. In contrast, LPA and S1P stimulate transient cell rounding and aggregation that is independent of EGFR and ERK, but dependent on the Rho effector p160 ROCK. Conclusion Thus, lysophospholipids regulate neural progenitor growth and morphology through distinct mechanisms. These findings establish human ES cell-derived NEP cells as a model system for studying the role of lysophospholipids in neural progenitors. PMID:19077254

  1. Peroxisome proliferator-activated receptor {alpha} agonist-induced down-regulation of hepatic glucocorticoid receptor expression in SD rats

    SciTech Connect

    Chen Xiang; Li Ming; Sun Weiping; Bi Yan; Cai Mengyin; Liang Hua; Yu Qiuqiong; He Xiaoying; Weng Jianping

    2008-04-18

    It was reported that glucocorticoid production was inhibited by fenofibrate through suppression of type-1 11{beta}-hydroxysteroid dehydrogenase gene expression in liver. The inhibition might be a negative-feedback regulation of glucocorticoid receptor (GR) activity by peroxisome proliferator-activated receptor alpha (PPAR{alpha}), which is quickly induced by glucocorticoid in the liver. However, it is not clear if GR expression is changed by fenofibrate-induced PPAR{alpha} activation. In this study, we tested this possibility in the liver of Sprague-Dawley rats. GR expression was reduced by fenofibrate in a time- and does-dependent manner. The inhibition was observed in liver, but not in fat and muscle. The corticosterone level in the blood was increased significantly by fenofibrate. These effects of fenofibrate were abolished by PPAR{alpha} inhibitor MK886, suggesting that fenofibrate activated through PPAR{alpha}. In conclusion, inhibition of GR expression may represent a new molecular mechanism for the negative feedback regulation of GR activity by PPAR{alpha}.

  2. Decreased expression of receptor tyrosine kinase of EphB1 protein in renal cell carcinomas

    PubMed Central

    Zhou, Shuigen; Wang, Longxin; Li, Guimei; Zhang, Zhengyu; Wang, Jiandong

    2014-01-01

    Receptors tyrosine kinase of Eph superfamily plays an important role in human cancers. We previously found that EphB1 subtype is down-regulated in gastric cancer, colorectal cancer and ovary serous carcinoma. Fore the more, the decreased expression of EphB1 is related to invasion and metastasis in cancers. Although EphB1 has been revealed as an important receptor in cancers, our understanding of its roles in renal cell carcinoma (RCC) is limited. In the present study, using specific anit-EphB1 polyclonal antibody and immunohistochemistry, we evaluated EphB1 protein expression levels in RCC specimens surgically resected from 82 patients (including 62 conventional clear-cell RCC, 10 papillary, and 10 chromophobic RCC cases). We found EphB1 protein is positively expressed in the epithelium of renal tubules. Decreased expression of EphB1 was found in all RCC carcinomas compared with expression in the normal epithelium of renal tubules. EphB1 protein moderately expressed in chromophobic RCC, weakly expressed in clear-cell RCC and negatively expressed in papillary RCC. Our results indicate that EphB1 may be involved in carcinogenesis of RCC, the molecular mechanisms of down-regulation of EphB1 including genetic and epigenetic alterations and the dedicated roles of EphB1 in occurrence and progress of RCC need to be explicated in next step. PMID:25120806

  3. Targeting the expression of integrin receptors in tumors

    NASA Astrophysics Data System (ADS)

    Bloch, Sharon; Liang, Kexian; Dorshow, Richard B.; Ye, Yunpeng; Achilefu, Samuel I.

    2004-06-01

    Expression of integrin αvβ3 is upregulated in a number of cancers including colon, pancreas, lung and breast. Additionally, αvβ3 integrin expression has been linked to tumor metastasis and targeting this cell surface protein could provide a viable approach to image and evaluate the metastatic potential of tumors. Accordingly, we evaluated the selective retention of some near infrared (NIR) fluorescent probes in nude mice bearing A549 lung cancer xenograft that express αvβ3 integrin. Our preliminary results indicate that a novel NIR probe designed to target this integrin selectively accumulated in A549 tumor while other non-integrin specific probes were not retained in the tumor. Blocking studies show that tumor uptake of the probe is mediated by αvβ3 integrin receptor.

  4. Combined modification of intracellular and extracellular loci on human gonadotropin-releasing hormone receptor provides a mechanism for enhanced expression.

    PubMed

    Maya-Núñez, G; Janovick, J A; Conn, P M

    2000-12-01

    The mammalian gonadotropin-releasing hormone (GnRH) receptor (GnRH-R) has been a therapeutic target for human and animal medicine. This receptor is a unique G-protein-coupled receptor that lacks the intracellular C-terminal domain commonly associated with this family. Development of highthrough put screens for agents active in humans has been hampered by low expression levels of the hGnRH-R in cellular models. Two sites have attracted the interest of laboratories studying regulation of expression. The chimeric addition of the C-terminal tail from catfish GnRH-R (cfGnRH-R) to the rat GnRH-R significantly augmented receptor expression in GH3 cells. In addition, rodent GnRH-R contains 327 amino acids, but cow, sheep, and human GnRH-R (hGnRH-R) contain 328 residues, the "additional" residue being a Lys 191. Deletion of Lys 191 (del 191) from the hGnRH-R resulted in increased receptor expression levels and decreased internalization rates in both COS-7 and HEK 293 cells. In this study, the combined effect of the addition of the C-tail from cfGnRH-R and deletion of the Lys 191 from the hGnRH-R was compared to expression of the wild-type (WT) or either alteration alone in a transient expression system using primate cells. The altered receptor (hGnRH-R[del 191]-C-tail) showed significantly increased receptor expression at the cell surface compared with the WT or either modification alone. The inositol phosphate response to stimulation was also significantly elevated in response to GnRH agonist. After treatment with a GnRH agonist, the altered receptors showed a slower internalization rate. The homologous steady-state regulation of the WT and the altered receptors was similar, although the response of the altered receptors was significantly decreased. These results suggest that the conformational change in the receptor as a result of the deletion of Lys 191 and the addition of the C-terminus tail substantially increased the steady-state receptor expression and decreased

  5. Activation of neurokinin-1 receptors up-regulates substance P and neurokinin-1 receptor expression in murine pancreatic acinar cells

    PubMed Central

    Koh, Yung-Hua; Moochhala, Shabbir; Bhatia, Madhav

    2012-01-01

    Abstract Acute pancreatitis (AP) has been associated with an up-regulation of substance P (SP) and neurokinin-1 receptor (NK1R) in the pancreas. Increased SP-NK1R interaction was suggested to be pro-inflammatory during AP. Previously, we showed that caerulein treatment increased SP/NK1R expression in mouse pancreatic acinar cells, but the effect of SP treatment was not evaluated. Pancreatic acinar cells were obtained from pancreas of male swiss mice (25–30 g). We measured mRNA expression of preprotachykinin-A (PPTA) and NK1R following treatment of SP (10−6M). SP treatment increased PPTA and NK1R expression in isolated pancreatic acinar cells, which was abolished by pretreatment of a selective NK1R antagonist, CP96,345. SP also time dependently increased protein expression of NK1R. Treatment of cells with a specific NK1R agonist, GR73,632, up-regulated SP protein levels in the cells. Using previously established concentrations, pre-treatment of pancreatic acinar cells with Gö6976 (10 nM), rottlerin (5 μM), PD98059 (30 μM), SP600125 (30 μM) or Bay11-7082 (30 μM) significantly inhibited up-regulation of SP and NK1R. These observations suggested that the PKC-ERK/JNK-NF-κB pathway is necessary for the modulation of expression levels. In comparison, pre-treatment of CP96,345 reversed gene expression in SP-induced cells, but not in caerulein-treated cells. Overall, the findings in this study suggested a possible auto-regulatory mechanism of SP/NK1R expression in mouse pancreatic acinar cells, via activation of NK1R. Elevated SP levels during AP might increase the occurrence of a positive feedback loop that contributes to abnormally high expression of SP and NK1R. PMID:22040127

  6. Human Gastroenteropancreatic Expression of Melatonin and Its Receptors MT1 and MT2

    PubMed Central

    Söderquist, Fanny; Hellström, Per M.; Cunningham, Janet L.

    2015-01-01

    Background and Aim The largest source of melatonin, according to animal studies, is the gastrointestinal (GI) tract but this is not yet thoroughly characterized in humans. This study aims to map the expression of melatonin and its two receptors in human GI tract and pancreas using microarray analysis and immunohistochemistry. Method Gene expression data from normal intestine and pancreas and inflamed colon tissue due to ulcerative colitis were analyzed for expression of enzymes relevant for serotonin and melatonin production and their receptors. Sections from paraffin-embedded normal tissue from 42 individuals, representing the different parts of the GI tract (n=39) and pancreas (n=3) were studied with immunohistochemistry using antibodies with specificity for melatonin, MT1 and MT2 receptors and serotonin. Results Enzymes needed for production of melatonin are expressed in both GI tract and pancreas tissue. Strong melatonin immunoreactivity (IR) was seen in enterochromaffin (EC) cells partially co-localized with serotonin IR. Melatonin IR was also seen in pancreas islets. MT1 and MT2 IR were both found in the intestinal epithelium, in the submucosal and myenteric plexus, and in vessels in the GI tract as well as in pancreatic islets. MT1 and MT2 IR was strongest in the epithelium of the large intestine. In the other cell types, both MT2 gene expression and IR were generally elevated compared to MT1. Strong MT2, IR was noted in EC cells but not MT1 IR. Changes in gene expression that may result in reduced levels of melatonin were seen in relation to inflammation. Conclusion Widespread gastroenteropancreatic expression of melatonin and its receptors in the GI tract and pancreas is in agreement with the multiple roles ascribed to melatonin, which include regulation of gastrointestinal motility, epithelial permeability as well as enteropancreatic cross-talk with plausible impact on metabolic control. PMID:25822611

  7. Pravastatin and C reactive protein modulate protease- activated receptor-1 expression in vitro blood platelets.

    PubMed

    Chu, L-X; Zhou, S-X; Yang, F; Qin, Y-Q; Liang, Z-S; Mo, C-G; Wang, X-D; Xie, J; He, L-P

    2016-01-01

    Protease-activated receptor-1 (PAR-1) plays an important role in mediating activation of human platelets by thrombin. However, mechanism of statin in ADP-induced platelet PAR-1 expression is also unknown. Aggregometry, flow cytometry, immunoblotting and ELISA were used to determine role of pravastatin participating in ADP-induced platelet activation and PAR-1 expression. ADP stimulation significantly increased PAR-1 expression on platelets. PAR-1 antagonist SCH-79797 inhibited platelet aggregation as well as decreased platelet P-selectin expression induced by ADP. CRP inhibited PAR-1 expression induced by ADP in a concentration-dependent manner. Pravastatin treatment reduced PAR-1 expression in a concentration-dependent manner. Combination treatment of CRP and Pravastatin significantly reduced platelet PAR-1 expression induced by ADP. By western-blot analysis, pravastatin treatment did not influence total PAR-1 after ADP treatment. CRP decreased platelet total PAR-1 expression induced by ADP. Pravastatin and CRP reduced TXB2 formation by ADP significantly. CRP decreased thrombin fragment F1+2 level with ADP treatment. Pravastatin, in contrast, did not influence F1+2 level. Upon treatment with Pravastatin reduced platelet LOX-1 expression induced by ADP. In conclusion, PAR-1 served as a critical mechanism to relay platelet activation process induced by ADP. CRP and pravastatin reduce PAR-1 expression in platelet by ADP pathway. PMID:26950455

  8. Downregulation of transferrin receptor surface expression by intracellular antibody

    SciTech Connect

    Peng Jilin; Wu Sha; Zhao Xiaoping; Wang Min; Li Wenhan; Shen Xin; Liu Jing; Lei Ping; Zhu Huifen; Shen Guanxin . E-mail: guanxin_shen@yahoo.com.cn

    2007-03-23

    To deplete cellular iron uptake, and consequently inhibit the proliferation of tumor cells, we attempt to block surface expression of transferrin receptor (TfR) by intracellular antibody technology. We constructed two expression plasmids (scFv-HAK and scFv-HA) coding for intracellular single-chain antibody against TfR with or without endoplasmic reticulum (ER) retention signal, respectively. Then they were transfected tumor cells MCF-7 by liposome. Applying RT-PCR, Western blotting, immunofluorescence microscopy and immunoelectron microscope experiments, we insure that scFv-HAK intrabody was successfully expressed and retained in ER contrasted to the secreted expression of scFv-HA. Flow cytometric analysis confirmed that the TfR surface expression was markedly decreased approximately 83.4 {+-} 2.5% in scFv-HAK transfected cells, while there was not significantly decrease in scFv-HA transfected cells. Further cell growth and apoptosis characteristics were evaluated by cell cycle analysis, nuclei staining and MTT assay. Results indicated that expression of scFv-HAK can dramatically induce cell cycle G1 phase arrest and apoptosis of tumor cells, and consequently significantly suppress proliferation of tumor cells compared with other control groups. For First time this study demonstrates the potential usage of anti-TfR scFv-intrabody as a growth inhibitor of TfR overexpressing tumors.

  9. Carnosine, nerve growth factor receptor and tyrosine hydroxylase expression during the ontogeny of the rat olfactory system.

    PubMed

    Biffo, S; Martí, E; Fasolo, A

    1992-01-01

    The localizations of carnosine, nerve growth factor (NGF) receptor and tyrosine hydroxylase (TH) were studied in the embryonic and postnatal rat olfactory bulb and epithelium by means of single- and double-immunostaining methods. Tyrosine hydroxylase ontogeny was also evaluated at the mRNA level by in situ hybridization. All these molecules were expressed in the olfactory bulb but with different developmental patterns and cellular localization: carnosine immunoreactivity is seen from embryonic day 17 in primary olfactory neurons scattered in the nasal cavity and in fibres projecting from them to the olfactory bulb. Nerve growth factor-receptor immunoreactivity associated with small glial-like cells is visible in some glomeruli starting from the second day of postnatal life. At postnatal day 10 NGF-receptor immunoreactivity is extended to all glomeruli. Periglomerular neurons expressing TH mRNA and protein are present prenatally and their number sharply increases during the early postnatal development. Double-staining methods show that TH and NGF-receptor immunoreactivity do not overlap in cell bodies and processes. In addition, NGF-receptor immunoreactivity is not colocalized with carnosine. These findings definitely exclude NGF-receptor expression in periglomerular and primary olfactory neurons, suggesting that at least part of NGF-receptor expression in the olfactory bulb is associated with glial cells. In addition, they provide the first immunohistochemical data on carnosine ontogeny and confirm at the mRNA level previous studies on the ontogeny of TH protein. PMID:1376608

  10. Anatomical and molecular consequences of Unilateral Naris Closure on two populations of olfactory sensory neurons expressing defined odorant receptors.

    PubMed

    Molinas, Adrien; Aoudé, Imad; Soubeyre, Vanessa; Tazir, Bassim; Cadiou, Hervé; Grosmaitre, Xavier

    2016-07-28

    Mammalian olfactory sensory neurons (OSNs), the primary elements of the olfactory system, are located in the olfactory epithelium lining the nasal cavity. Exposed to the environment, their lifespan is short. Consequently, OSNs are regularly regenerated and several reports show that activity strongly modulates their development and regeneration: the peripheral olfactory system can adjust to the amount of stimulus through compensatory mechanisms. Unilateral naris occlusion (UNO) was frequently used to investigate this mechanism at the entire epithelium level. However, there is little data regarding the effects of UNO at the cellular level, especially on individual neuronal populations expressing a defined odorant receptor. Here, using UNO during the first three postnatal weeks, we analyzed the anatomical and molecular consequences of sensory deprivation in OSNs populations expressing the MOR23 and M71 receptors. The density of MOR23-expressing neurons is decreased in the closed side while UNO does not affect the density of M71-expressing neurons. Using Real Time qPCR on isolated neurons, we observed that UNO modulates the transcript levels for transduction pathway proteins (odorant receptors, CNGA2, PDE1c). The transcripts modulated by UNO will differ between populations depending on the receptor expressed. These results suggest that sensory deprivation will have different effects on different OSNs' populations. As a consequence, early experience will shape the functional properties of OSNs differently depending on the type of odorant receptor they express. PMID:27189720

  11. Abnormal gene expression of proinflammatory cytokines and their membrane-bound receptors in the lymphocytes of depressed patients.

    PubMed

    Rizavi, Hooriyah S; Ren, Xinguo; Zhang, Hui; Bhaumik, Runa; Pandey, Ghanshyam N

    2016-06-30

    Abnormalities of protein levels of proinflammatory cytokines and their soluble receptors have been reported in plasma of depressed patients. In this study, we examined the role of cytokines and their membrane-bound receptors in major depressive disorder (MDD). We determined the protein and mRNA expression of proinflammatory cytokines, interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, and mRNA expression of their membrane-bound receptors in the lymphocytes from 31 hospitalized MDD patients and 30 non-hospitalized normal control (NC) subjects. The subjects were diagnosed according to DSM-IV criteria. Protein levels of cytokines were determined by ELISA, and mRNA levels in lymphocytes were determined by the qPCR method. We found that the mean mRNA levels of the proinflammatory cytokines IL-1β, IL-6, TNF-α, their receptors, TNFR1, TNFR2, IL-1R1 and the antagonist IL-1RA were significantly increased in the lymphocytes of MDD patients compared with NC. No significant differences in the lymphocyte mRNA levels of IL-1R2, IL-6R, and Gp130 were observed between MDD patients and NC. These studies suggest abnormal gene expression of these cytokines and their membrane-bound receptors in the lymphocytes of MDD patients, and that their mRNA expression levels in the lymphocytes could be a useful biomarker for depression. PMID:27138824

  12. Potential clinical relevance of Eph receptors and ephrin ligands expressed in prostate carcinoma cell lines.

    PubMed

    Fox, Brian P; Tabone, Christopher J; Kandpal, Raj P

    2006-04-21

    The family of Eph and ephrin receptors is involved in a variety of functions in normal cells, and the alterations in their expression profiles have been observed in several cancers. We have compared the transcripts for Eph receptors and ephrin ligands in cell lines established from normal prostate epithelium and several carcinoma cell lines isolated from prostate tumors of varying degree of metastasis. These cell lines included NPTX, CTPX, LNCaP, DU145, PC-3, and PC-3ML. The cell lines displayed characteristic pattern of expression for specific Eph receptors and ephrin ligands, thus allowing identification of Eph receptor signatures for a particular cell line. The sensitivity of these transcripts to genome methylation is also investigated by treating the cells with 5-aza-2'-deoxycytidine. The comparison of expression profiles revealed that normal prostate and primary prostate tumor cell lines differ in the expression of EphA3, EphB3, and ephrin A3 that are over-expressed in normal prostate. Furthermore, the transcript levels for EphA1 decrease progressively from normal prostate to primary prostate tumor cell line and metastatic tumor cells. A converse relationship was observed for ephrin B2. The treatment of cells with 5-aza-2'-deoxycytidine revealed the sensitivity of EphA3, EphA10, EphB3, and EphB6 to methylation status of genomic DNA. The utility of methylation specific PCR to identify prostate tumor cells and the importance of specific Eph receptors and ephrin ligands in initiation and progression of prostate tumor are discussed. PMID:16516143

  13. Regulation of p53 expression, phosphorylation and sub-cellular localisation by a G-protein coupled receptor

    PubMed Central

    Solyakov, Lev; Sayan, Emre; Riley, Joan; Pointon, Amy; Tobin, Andrew B

    2009-01-01

    G-protein coupled receptors (GPCRs) have been extremely successful drug targets for a multitude of diseases from heart failure to depression. This super-family of cell surface receptors have not, however, been widely considered as a viable target in cancer treatment. In the current study we demonstrate that a classical Gq/11-coupled GPCR, the M3-muscarinic receptor, was able to regulate apoptosis via receptors that are endogenously expressed in the human neuroblastoma cell line SH-SY5Y and when ectopically expressed in Chinese hamster ovary (CHO) cells. Stimulation of the M3-muscarinic receptor was shown to inhibit the ability of the DNA-damaging chemotherapeutic agent, etoposide, from mediating apoptosis. This protective response in CHO cells correlated with the ability of the receptor to regulate the expression levels of p53. In contrast, stimulation of endogenous muscarinic receptors in SH-SY5Y cells did not regulate p53 expression but rather was able to inhibit p53 translocation to the mitochondria and p53 phosphorylation at serine 15 and 37. This study suggests the possibility that a GPCR can regulate the apoptotic properties of a chemotherapeutic DNA-damaging agent by regulating the expression, sub-cellular trafficking and modification of p53 in a manner that is in part dependent on the cell type. PMID:19648965

  14. Evidence for estrogen receptor expression in germ cell and somatic cell subpopulations in the ovary of the newly hatched chicken.

    PubMed

    Méndez, M C; Chávez, B; Echeverría, O; Vilchis, F; Vázquez Nin, G H; Pedernera, E

    1999-10-01

    Estrogens are involved in the gonadal morphogenesis of vertebrates, and almost all hormonal effects of 17beta-estradiol are mediated through specific receptors. At the time of sexual differentiation in the chicken, or even before, there is evidence of the presence of estrogen receptors and the secretion of 17beta-estradiol. However, no information is available regarding the cellular types that express the estrogen receptor in the immature chick ovary. The present study analyzes estrogen receptor expression in germ and somatic cells of the ovary in the newly hatched chicken. Highly purified cell subpopulations of germ and somatic cells were evaluated for specific 17beta-estradiol nuclear binding. In addition, the estrogen receptor was localized at the ultrastructural level by the immunogold technique. Finally, reverse transcription and polymerase chain reaction procedures detected a steady-state level of mRNA for the estrogen receptor. Somatic cells including typical steroidogenic cells showed specific 17beta-estradiol nuclear binding, displayed the estrogen receptor, and possessed estrogen receptor transcripts. The same result was observed in primary oocytes, together with the ultrastructural localization of estrogen receptor in extended chromatin filaments. Our experimental data support the hypothesis that estrogens are involved in the function of somatic and germ cells subpopulations in the immature chicken ovary. PMID:10555548

  15. Expression of the Somatostatin Receptor Subtype 4 in Intact and Inflamed Pulmonary Tissues

    PubMed Central

    Varecza, Zoltán; Elekes, Krisztián; László, Terézia; Perkecz, Anikó; Pintér, Erika; Sándor, Zoltán; Szolcsányi, János; Keszthelyi, Dániel; Szabó, Árpád; Sándor, Katalin; Molnár, Tamás F.; Szántó, Zalán; Pongrácz, Judit E.; Helyes, Zsuzsanna

    2009-01-01

    Somatostatin released from capsaicin-sensitive sensory nerves of the lung during endotoxin-induced murine pneumonitis inhibits inflammation and hyperresponsiveness, presumably via somatostatin receptor subtype 4 (sst4). The goal of the present study was to identify sst4 receptors in mouse and human lungs and to reveal its inflammation-induced alterations with real-time quantitative PCR, Western blot, and immunohistochemistry. In non-inflamed mouse and human lungs, mRNA expression and immunolocalization of sst4 are very similar. They are present on bronchial epithelial, vascular endothelial, and smooth-muscle cells. The sst4 receptor protein in the mouse lung significantly increases 24 hr after intranasal endotoxin administration as well as in response to 3 months of whole-body cigarette smoke exposure, owing to the infiltrating sst4-positivite mononuclear cells and neutrophils. In the chronically inflamed human lung, the large number of activated macrophages markedly elevate sst4 mRNA levels, although there is no change in acute purulent pneumonia, in which granulocytes accumulate. Despite mouse granulocytes, human neutrophils do not show sst4 immunopositivity. We provide the first evidence for the expression, localization, and inflammation-induced alterations of sst4 receptors in murine and human lungs. Inasmuch as tissue distribution of this receptor is highly similar, extrapolation of murine experimental results to human conditions might be possible. (J Histochem Cytochem 57:1127–1137, 2009) PMID:19687471

  16. Differential expression of TRAIL and its receptors relative to calcification in AAA

    SciTech Connect

    Liu, Xun . E-mail: mpscrs@bath.ac.uk; Winrow, Vivienne R.; Horrocks, Michael; Stevens, Cliff R.

    2007-06-22

    Abdominal aortic aneurysm (AAA) is commonly associated with atherosclerosis. Human AAA tissue displays cells undergoing all stages of apoptosis. Tumour necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) induces apoptosis in tumour cells but not in normal cells. It has death receptors and decoy receptors. An inhibitor of TRAIL, osteoprotegerin (OPG), is involved in osteogenesis and vascular calcification. We investigated TRAIL and its receptors in AAA compared within normal aorta (NA). Both qualitative and quantitative analyses of calcification in AAA walls were determined using Von Kossa staining and pre-operation computer tomography (CT) scans. There was a significant difference in calcification level at different locations in the AAA wall (p < 0.05). Apoptosis was confirmed in AAA by TUNEL assay. A significant difference in TRAIL and its receptor expression was observed between normal aortae and AAA (p < 0.05). Significant differences were also observed between tissues displaying different extents of calcification for TRAIL mRNA (p < 0.05) by RT-PCR examination and OPG protein (p < 0.01) by protein blotting examination. We propose that this pattern of expression of TRAIL and its receptors may contribute to AAA formation and calcification in the AAA wall.

  17. Simvastatin enhances bone morphogenetic protein receptor type II expression

    SciTech Connect

    Hu Hong; Sung, Arthur; Zhao, Guohua; Shi, Lingfang; Qiu Daoming; Nishimura, Toshihiko; Kao, Peter N. . E-mail: peterkao@stanford.edu

    2006-01-06

    Statins confer therapeutic benefits in systemic and pulmonary vascular diseases. Bone morphogenetic protein (BMP) receptors serve essential signaling functions in cardiovascular development and skeletal morphogenesis. Mutations in BMP receptor type II (BMPR2) are associated with human familial and idiopathic pulmonary arterial hypertension, and pathologic neointimal proliferation of vascular endothelial and smooth muscle cells within small pulmonary arteries. In severe experimental pulmonary hypertension, simvastatin reversed disease and conferred a 100% survival advantage. Here, modulation of BMPR2 gene expression by simvastatin is characterized in human embryonic kidney (HEK) 293T, pulmonary artery smooth muscle, and lung microvascular endothelial cells (HLMVECs). A 1.4 kb BMPR2 promoter containing Egr-1 binding sites confers reporter gene activation in 293T cells which is partially inhibited by simvastatin. Simvastatin enhances steady-state BMPR2 mRNA and protein expression in HLMVEC, through posttranscriptional mRNA stabilization. Simvastatin induction of BMPR2 expression may improve BMP-BMPR2 signaling thereby enhancing endothelial differentiation and function.

  18. Targeting receptor for advanced glycation end products (RAGE) expression induces apoptosis and inhibits prostate tumor growth

    SciTech Connect

    Elangovan, Indira; Thirugnanam, Sivasakthivel; Chen, Aoshuang; Zheng, Guoxing; Bosland, Maarten C.; Kajdacsy-Balla, Andre; Gnanasekar, Munirathinam

    2012-01-27

    Highlights: Black-Right-Pointing-Pointer Targeting RAGE by RNAi induces apoptosis in prostate cancer cells. Black-Right-Pointing-Pointer Silencing RAGE expression abrogates rHMGB1 mediated cell proliferation. Black-Right-Pointing-Pointer Down regulation of RAGE by RNAi inhibits PSA secretion of prostate cancer cells. Black-Right-Pointing-Pointer Knock down of RAGE abrogates prostate tumor growth in vivo. Black-Right-Pointing-Pointer Disruption of RAGE expression in prostate tumor activates death receptors. -- Abstract: Expression of receptor for advanced glycation end products (RAGE) plays a key role in the progression of prostate cancer. However, the therapeutic potential of targeting RAGE expression in prostate cancer is not yet evaluated. Therefore in this study, we have investigated the effects of silencing the expression of RAGE by RNAi approach both in vitro and in vivo. The results of this study showed that down regulation of RAGE expression by RNAi inhibited the cell proliferation of androgen-dependent (LNCaP) and androgen-independent (DU-145) prostate cancer cells. Furthermore, targeting RAGE expression resulted in apoptotic elimination of these prostate cancer cells by activation of caspase-8 and caspase-3 death signaling. Of note, the levels of prostate specific antigen (PSA) were also reduced in LNCaP cells transfected with RAGE RNAi constructs. Importantly, the RAGE RNAi constructs when administered in nude mice bearing prostate tumors, inhibited the tumor growth by targeting the expression of RAGE, and its physiological ligand, HMGB1 and by up regulating death receptors DR4 and DR5 expression. Collectively, the results of this study for the first time show that targeting RAGE by RNAi may be a promising alternative therapeutic strategy for treating prostate cancer.

  19. Expression of FSH receptor in the hamster ovary during perinatal development

    PubMed Central

    Chakraborty, Prabuddha; Roy, Shyamal K.

    2014-01-01

    FSH plays an important role in ovarian follicular development, and it functions via the G-protein coupled FSH receptor. The objectives of the present study were to determine if full-length FSHR mRNA and corresponding protein were expressed in fetal through postnatal hamster ovaries to explain the FSH-induced primordial follicle formation, and if FSH or estrogen (E) would affect the expression. A full-length and two alternately spliced FSHR transcripts were expressed from E14 through P20. The level of the full-length FSHR mRNA increased markedly through P7 before stabilizing at a lower level with the formation and activation of primordial follicles. A predicted 87kDa FSHR protein band was detected in fetal through P4 ovaries, but additional bands appeared as ovary developed. FSHR immunosignal was present in undifferentiated somatic cells and oocytes in early postnatal ovaries, but was granulosa cells specific after follicles formed. Both eCG and E significantly up-regulated full-length FSHR mRNA levels. Therefore, FSHR is expressed in the hamster ovary from the fetal life to account for FSH-induced primordial follicle formation and cAMP production. Further, FSH or E regulates the receptor expression. PMID:25462586

  20. Functional characterization of an allatotropin receptor expressed in the corpora allata of mosquitoes

    PubMed Central

    Nouzova, Marcela; Brockhoff, Anne; Mayoral, Jaime G.; Goodwin, Marianne; Meyerhof, Wolfgang; Noriega, Fernando G.

    2011-01-01

    Allatotropin is an insect neuropeptide with pleiotropic actions on a variety of different tissues. In the present work we describe the identification, cloning and functional and molecular characterization of an Aedes aegypti allatotropin receptor (AeATr) and provide a detailed quantitative study of the expression of the AeATr gene in the adult mosquito. Analysis of the tissue distribution of AeATr mRNA in adult female revealed high transcript levels in the nervous system (brain, abdominal, thoracic and ventral ganglia), corpora allata-corpora cardiaca complex and ovary. The receptor is also expressed in heart, hindgut and male testis and accessory glands. Separation of the corpora allata (CA) and corpora cardiaca followed by analysis of gene expression in the isolated glands revealed expression of the AeATr primarily in the CA. In the female CA, the AeATr mRNA levels were low in the early pupae, started increasing 6 hours before adult eclosion and reached a maximum 24 hours after female emergence. Blood feeding resulted in a decrease in transcript levels. The pattern of changes of AeATr mRNA resembles the changes in JH biosynthesis. Fluorometric Imaging Plate Reader recordings of calcium transients in HEK293 cells expressing the AeATr showed a selective response to A. aegypti allatotropin stimulation in the low nanomolar concentration range. Our studies suggest that the AeATr play a role in the regulation of JH synthesis in mosquitoes. PMID:21839791

  1. Estrogen Receptor Alpha Distribution and Expression in the Social Neural Network of Monogamous and Polygynous Peromyscus

    PubMed Central

    Cushing, Bruce S.

    2016-01-01

    In microtine and dwarf hamsters low levels of estrogen receptor alpha (ERα) in the bed nucleus of the stria terminalis (BST) and medial amygdala (MeA) play a critical role in the expression of social monogamy in males, which is characterized by high levels of affiliation and low levels of aggression. In contrast, monogamous Peromyscus males display high levels of aggression and affiliative behavior with high levels of testosterone and aromatase activity. Suggesting the hypothesis that in Peromyscus ERα expression will be positively correlated with high levels of male prosocial behavior and aggression. ERα expression was compared within the social neural network, including the posterior medial BST, MeA posterodorsal, medial preoptic area (MPOA), ventromedial hypothalamus (VMH), and arcuate nucleus in two monogamous species, P. californicus and P. polionotus, and two polygynous species, P. leucopus and P. maniculatus. The results supported the prediction, with male P. polionotus and P. californicus expressing higher levels of ERα in the BST than their polygynous counter parts, and ERα expression was sexually dimorphic in the polygynous species, with females expressing significantly more than males in the BST in both polygynous species and in the MeA in P. leucopus. Peromyscus ERα expression also differed from rats, mice and microtines as in neither the MPOA nor the VMH was ERα sexually dimorphic. The results supported the hypothesis that higher levels of ERα are associated with monogamy in Peromyscus and that differential expression of ERα occurs in the same regions of the brains regardless of whether high or low expression is associated with social monogamy. Also discussed are possible mechanisms regulating this differential relationship. PMID:26959827

  2. Gene Expression Switching of Receptor Subunits in Human Brain Development

    PubMed Central

    Bar-Shira, Ossnat; Maor, Ronnie; Chechik, Gal

    2015-01-01

    Synaptic receptors in the human brain consist of multiple protein subunits, many of which have multiple variants, coded by different genes, and are differentially expressed across brain regions and developmental stages. The brain can tune the electrophysiological properties of synapses to regulate plasticity and information processing by switching from one protein variant to another. Such condition-dependent variant switch during development has been demonstrated in several neurotransmitter systems including NMDA and GABA. Here we systematically detect pairs of receptor-subunit variants that switch during the lifetime of the human brain by analyzing postmortem expression data collected in a population of donors at various ages and brain regions measured using microarray and RNA-seq. To further detect variant pairs that co-vary across subjects, we present a method to quantify age-corrected expression correlation in face of strong temporal trends. This is achieved by computing the correlations in the residual expression beyond a cubic-spline model of the population temporal trend, and can be seen as a nonlinear version of partial correlations. Using these methods, we detect multiple new pairs of context dependent variants. For instance, we find a switch from GLRA2 to GLRA3 that differs from the known switch in the rat. We also detect an early switch from HTR1A to HTR5A whose trends are negatively correlated and find that their age-corrected expression is strongly positively correlated. Finally, we observe that GRIN2B switch to GRIN2A occurs mostly during embryonic development, presumably earlier than observed in rodents. These results provide a systematic map of developmental switching in the neurotransmitter systems of the human brain. PMID:26636753

  3. Low renal mineralocorticoid receptor expression at birth contributes to partial aldosterone resistance in neonates

    PubMed Central

    Martinerie, Laetitia; Viengchareun, Say; Delezoïde, Anne-Lise; Jaubert, Francis; Sinico, Martine; Prevot, Sophie; Boileau, Pascal; Meduri, Géri; Lombès, Marc

    2009-01-01

    The human neonatal period is characterized by renal immaturity with impaired capacity to regulate water and sodium homeostasis, resembling partial aldosterone resistance. Since aldosterone effects are mediated by the mineralocorticoid receptor (MR), we postulated that this hormonal unresponsiveness could be related to low MR expression in the distal nephron. We measured aldosterone and renin levels in umbilical cord blood of healthy newborns. We used qPCR and immunohistochemistry to analyze the expression of MR and key players of the mineralocorticoid signaling pathway, during human and mouse renal development. High aldosterone and renin levels were found at birth. MR mRNA was detected in mouse kidney at day 16 postcoitum (E16), peaking at E18, but its expression was surprisingly very low at birth, rising progressively afterwards. Similar biphasic temporal expression was observed during human renal embryogenesis, with a transient expression between 15 and 24 weeks of gestation but an undetectable immunoreactive MR in late gestational and neonatal kidneys. This cyclic MR expression was tightly correlated with the evolution of the 11β–hydroxysteroid dehydrogenase type 2 and the epithelial sodium channel α-subunit. In contrast, glucocorticoid and vasopressin receptors, and aquaporin 2 followed a progressive and sustained evolution during renal maturation. Our study provides first evidence for a low renal MR expression level at birth, despite high aldosterone levels, which could account for compromised postnatal sodium handling. Elucidation of regulatory mechanisms governing MR expression should lead to new strategies for the management of sodium waste in preterms and neonates. PMID:19477942

  4. Unexpected Novel Relational Links Uncovered by Extensive Developmental Profiling of Nuclear Receptor Expression

    PubMed Central

    Tavares, Raquel; Sachs, Laurent; Chaumot, Arnaud; Bardet, Pierre-Luc; Escrivà, Héctor; Duffraisse, Maryline; Marchand, Oriane; Safi, Rachid; Thisse, Christine; Laudet, Vincent

    2007-01-01

    Nuclear receptors (NRs) are transcription factors that are implicated in several biological processes such as embryonic development, homeostasis, and metabolic diseases. To study the role of NRs in development, it is critically important to know when and where individual genes are expressed. Although systematic expression studies using reverse transcriptase PCR and/or DNA microarrays have been performed in classical model systems such as Drosophila and mouse, no systematic atlas describing NR involvement during embryonic development on a global scale has been assembled. Adopting a systems biology approach, we conducted a systematic analysis of the dynamic spatiotemporal expression of all NR genes as well as their main transcriptional coregulators during zebrafish development (101 genes) using whole-mount in situ hybridization. This extensive dataset establishes overlapping expression patterns among NRs and coregulators, indicating hierarchical transcriptional networks. This complete developmental profiling provides an unprecedented examination of expression of NRs during embryogenesis, uncovering their potential function during central nervous system and retina formation. Moreover, our study reveals that tissue specificity of hormone action is conferred more by the receptors than by their coregulators. Finally, further evolutionary analyses of this global resource led us to propose that neofunctionalization of duplicated genes occurs at the levels of both protein sequence and RNA expression patterns. Altogether, this expression database of NRs provides novel routes for leading investigation into the biological function of each individual NR as well as for the study of their combinatorial regulatory circuitry within the superfamily. PMID:17997606

  5. The chemosensory receptors of codling moth Cydia pomonella–expression in larvae and adults

    PubMed Central

    Walker, William B.; Gonzalez, Francisco; Garczynski, Stephen F.; Witzgall, Peter

    2016-01-01

    Olfaction and gustation play critical roles in the life history of insects, mediating vital behaviors such as food, mate and host seeking. Chemosensory receptor proteins, including odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) function to interface the insect with its chemical environment. Codling moth, Cydia pomonella, is a worldwide pest of apple, pear and walnut, and behavior-modifying semiochemicals are used for environmentally safe control. We produced an Illumina-based transcriptome from antennae of males and females as well as neonate head tissue, affording a qualitative and quantitative analysis of the codling moth chemosensory receptor repertoire. We identified 58 ORs, 20 GRs and 21 IRs, and provide a revised nomenclature that is consistent with homologous sequences in related species. Importantly, we have identified several OR transcripts displaying sex-biased expression in adults, as well as larval-enriched transcripts. Our analyses have expanded annotations of the chemosensory receptor gene families, and provide first-time transcript abundance estimates for codling moth. The results presented here provide a strong foundation for future work on codling moth behavioral physiology and ecology at the molecular level, and may lead to the development of more precise biorational control strategies. PMID:27006164

  6. The chemosensory receptors of codling moth Cydia pomonella-expression in larvae and adults.

    PubMed

    Walker, William B; Gonzalez, Francisco; Garczynski, Stephen F; Witzgall, Peter

    2016-01-01

    Olfaction and gustation play critical roles in the life history of insects, mediating vital behaviors such as food, mate and host seeking. Chemosensory receptor proteins, including odorant receptors (ORs), gustatory receptors (GRs) and ionotropic receptors (IRs) function to interface the insect with its chemical environment. Codling moth, Cydia pomonella, is a worldwide pest of apple, pear and walnut, and behavior-modifying semiochemicals are used for environmentally safe control. We produced an Illumina-based transcriptome from antennae of males and females as well as neonate head tissue, affording a qualitative and quantitative analysis of the codling moth chemosensory receptor repertoire. We identified 58 ORs, 20 GRs and 21 IRs, and provide a revised nomenclature that is consistent with homologous sequences in related species. Importantly, we have identified several OR transcripts displaying sex-biased expression in adults, as well as larval-enriched transcripts. Our analyses have expanded annotations of the chemosensory receptor gene families, and provide first-time transcript abundance estimates for codling moth. The results presented here provide a strong foundation for future work on codling moth behavioral physiology and ecology at the molecular level, and may lead to the development of more precise biorational control strategies. PMID:27006164

  7. The adaptor 3BP2 is required for KIT receptor expression and human mast cell survival

    PubMed Central

    Ainsua-Enrich, Erola; Serrano-Candelas, Eva; Álvarez-Errico, Damiana; Picado, César; Sayós, Joan; Rivera, Juan; Martín, Margarita

    2015-01-01

    3BP2 is a cytoplasmic adaptor protein that acts as a positive regulator in mast cell FcεRI-dependent signaling. The KIT receptor whose ligand is the stem cell factor (SCF) is necessary for mast cell development, proliferation and survival as well as for optimal IgE-dependent signal. Activating mutations in KIT have been associated with several diseases including mastocytosis. In the present work, we found that 3BP2 silencing impairs KIT signaling pathways, thus affecting PI3K and MAP kinase pathways in human mast cells from HMC-1, LAD2 (human mast cell lines) and CD34+-derived mast cells. Unexpectedly, silencing of 3BP2 reduces KIT expression in normal human mast cells as well as in HMC-1 cells where KIT is mutated, thus increasing cellular apoptosis and caspase 3/7 activity. 3BP2 silencing reduces KIT transcription expression levels. Interestingly, 3BP2 silencing decreased MITF expression, a transcription factor involved in KIT expression. Reconstitution of 3BP2 in knockdown cells leads to reversal of KIT expression as well as survival phenotype. Accordingly MITF reconstitution enhances KIT expression levels in 3BP2 silenced cells. Moreover, downregulation of KIT expression by miRNA221 overexpression or the proteasome inhibitor bortezomib also reduced 3BP2 and MITF expression. Furthermore, KIT tyrosine activity inhibition reduced 3BP2 and MITF expression, demonstrating again a tight and reciprocal relationship between these molecules. Taken together, our results show that 3BP2 regulates human mast cell survival and participates in KIT-mediated signal transduction by directly controlling KIT receptor expression, suggesting its potential as a therapeutic target in mast cell-mediated inflammatory diseases and deregulated KIT disorders. PMID:25810396

  8. Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes.

    PubMed

    Jiang, Yan J; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M; Holleran, Walter M; Grunfeld, Carl; Feingold, Kenneth R

    2009-07-10

    ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed

  9. Expression of NGF family and their receptors in gastric carcinoma: A cDNA microarray study

    PubMed Central

    Du, Jian-Jun; Dou, Ke-Feng; Peng, Shu-You; Qian, Bing-Zhi; Xiao, Hua-Sheng; Liu, Feng; Wang, Wei-Zhong; Guan, Wen-Xian; Gao, Zhi-Qing; Liu, Ying-Bin; Han, Ze-Guang

    2003-01-01

    AIM: To investigate the expression of NGF family and their receptors in gastric carcinoma and normal gastric mucosa, and to elucidate their effects on gastric carcinoma. METHODS: RNA of gastric cancer tissues and normal gastric tissues was respectively isolated and mRNA was purified. Probes of both mRNA reverse transcription product cDNAs labled with α-33P dATP were respectively hybridized with Atlas Array membrane where NGF and their family genes were spotted on. Hybridized signal images were scanned on phosphor screen with ImageQuant 5.1 software after hybridization. Normalized values on spots were analyzed with ArrayVersion 5.0 software. Differential expression of NGF family and their receptors mRNA was confirmed between hybridized Atlas Array membranes of gastric cancer tissues and normal gastric mucosa, then their effects on gastric carcinoma were investigated. RESULTS: Hybridization signal images on Atlas Array membrane appeared in a lower level of nonspecific hybridization. Both of NGF family and their receptors Trk family mRNA were expressed in gastric cancer and normal gastric mucosa. But adversely up-regulated expression in other tissues and organs. NGF, BDGF, NT-3, NT-4/5, NT-6 and TrkA, B and C were down-regulated simultaneously in gastric carcinoma in comparison with normal gastric mucosa. Degrees of down-regulation in NGF family were greater than those in their receptors Trk family. Down-regulation of NT-3 and BDGF was the most significant, and TrkC down-regulation level was the lowest in receptors Trk family. CONCLUSION: Down-regulated expression of NGF family and their receptors Trk family mRNA in gastric cancer is confirmed. NGF family and their receptors Trk family probably play a unique role in gastric cancer cell apoptosis by a novel Ras or Raf signal transduction pathway. Their synchronous effects are closely associated with occurrence and development of gastric carcinoma induced by reduction of signal transduction of programmed cell death

  10. [Expression of Toll-like receptors in human bone marrow mesenchymal stem cells].

    PubMed

    He, Xiao-Xia; Bai, Hai; Yang, Guo-Rong; Xue, Yong-Jie; Su, Ya-Nan

    2009-06-01

    The aim of this study was to explore the characteristics of Toll-like receptor expression in mesenchymal stem cells derived from bone marrow of healthy donor (BM-MSCs). BM-MSCs were isolated from bone marrow of healthy donor by Ficoll method. Expressions of CD34, CD45, HLA-DR, CD44 and CD71 in BM-MSCs were detected by flow cytometry. CD71 in BM-MSCs was assayed by immunocytochemistry. The adipocyte and osteoblast induction of BM-MSCs were detected by alizarin red stain and oil red stain respectively. TLR 1 - 10 mRNA levels in BM-MSCs were evaluated by semiquantitative RT-PCR. The results showed that expressions of CD34, CD45 and HLA-DR in BM-MSC were negative while the expressions of CD44 and CD71 were positive. CD71 in BM-MSCs was positive. After induced by osteoblast and adipocyte inductor, BM-MSCs were positive for alizarin red staining and oil red staining respectively. All of TLR 1 - 10 mRNA were found in BM-MSCs with high expression levels of TLR2, TLR3, TLR4, TLR7, TLR8, TLR9 and low expression levels of TLR1, TLR5, TLR6, TLR10. In conclusion, different levels of TLR 1 - 10 mRNA were expressed in BM-MSCs of healthy donor. PMID:19549390

  11. Bisphenol a influences blastocyst implantation via regulating integrin β3 and trophinin expression levels

    PubMed Central

    Pan, Xiaoyan; Wang, Xiyan; Wang, Zhengchao; Wang, Xuenan; Dou, Zhaohua; Li, Zhixin

    2015-01-01

    Objective: This study is to investigate effects of bisphenol A (BPA) on the blastocyst implantation in endometrium. Methods: Pregnant mice were orally administered with BPA. Implantation sites were examined, and serum estrogen level was assayed with ELISA. Protein expression levels were detected with immunohistochemistry and immunofluorescence staining. Results: High doses (400 and 600 mg/kg/day) of BPA remarkably reduced the implantation sites in the pregnant mice. No significant differences were observed in the serum estrogen level across the groups. Moreover, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression level of endometrial estrogen receptor α (ERα) in the pregnant mice. In addition, high doses (400 and 600 mg/kg/day) of BPA significantly declined the expression levels of integrin β3 and trophinin in the endometrium and blastocysts. Conclusion: BPA declines ERα expression in endometrium, and inhibits adhesion protein expression in endometrium and blastocysts, causing the adhesion failure of blastocyst implantation. PMID:26884915

  12. Characterization, expression analysis and localization pattern of toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes in grass carp Ctenopharyngodon idella.

    PubMed

    He, L B; Wang, H; Luo, L F; Jiang, S H; Liu, L Y; Li, Y M; Huang, R; Liao, L J; Zhu, Z Y; Wang, Y P

    2016-08-01

    In this study, the toll-like receptor 1 (tlr1) and toll-like receptor 2 (tlr2) genes of grass carp Ctenopharyngodon idella were cloned and characterized. tlr1 and tlr2 were found to be highly expressed in immune system organs such as spleen, middle kidney and heart kidney. The expression level of tlr1 and tlr2 was found to be up-regulated at the later stage of viral challenge process. Moreover, subcellular localization indicated that Tlr1 and Tlr2 shared similar localization pattern and both of them may locate in the plasma membrane of transfected cells. PMID:27221024

  13. Expression and functional roles of estrogen receptor GPR30 in human intervertebral disc.

    PubMed

    Wei, Aiqun; Shen, Bojiang; Williams, Lisa A; Bhargav, Divya; Yan, Feng; Chong, Beng H; Diwan, Ashish D

    2016-04-01

    Estrogen withdrawal, a characteristic of female aging, is associated with age-related intervertebral disc (IVD) degeneration. The function of estrogen is mediated by two classic nuclear receptors, estrogen receptor (ER)-α and -β, and a membrane bound G-protein-coupled receptor 30 (GPR30). To date, the expression and function of GPR30 in human spine is poorly understood. This study aimed to evaluate GPR30 expression in IVD, and its role in estrogen-related regulation of proliferation and apoptosis of disc nucleus pulposus (NP) cells. GPR30 expression was examined in 30 human adult NP and 9 fetal IVD. Results showed that GPR30 was expressed in NP cells at both mRNA and protein levels. In human fetal IVD, GPR30 protein was expressed in the NP at 12-14 weeks gestation, but was undetectable at 8-11 weeks. The effect of 17β-estradiol (E2) on GPR30-mediated proliferation and interleukin-1β (IL-1β)-induced apoptosis of NP cells was investigated. Cultured NP cells were treated with or without E2, GPR30 antagonist G36, and ER antagonist ICI 182,780. NP cell viability was tested by MTS assay. Apoptosis was determined by flow cytometry using fluorescence labeled annexin-V, TUNEL assay and immumnocytochemical staining of activated caspase-3. E2 enhanced cell proliferation and prevented IL-1β-induced cell death, but the effect was partially blocked by G36 and completely abrogated by a combination of ICI 182,780 and G36. This study demonstrates that GPR30 is expressed in human IVD to transmit signals triggering E2-induced NP cell proliferation and protecting against IL-1β-induced apoptosis. The effects of E2 on NP cells require both GPR30 and classic estrogen receptors. PMID:26815911

  14. Platelets deficient in glycoprotein I have normal Fc receptor expression.

    PubMed

    Pfueller, S L; de Rosbo, N K; Bilston, R A

    1984-04-01

    Platelet glycoprotein I (GPI) is known to be required for the interaction of platelets with ristocetin and factor VIII:von Willebrand factor (VIII:vWf). However, its role as Fc receptor is not clear. Some studies have shown that enzymatic removal of GPI destroys the ability of platelets to react with VIII:vWf but not their ability to bind Ig G (IgG). Others have shown that IgG immune complexes which block the Fc receptor also inhibit VIII:vWf interaction with platelets. This subject has been re-examined by testing the ability of platelets with reduced amounts of GPI to aggregate and undergo the release reaction in response to stimuli which act at the platelet Fc receptor. Platelets from two patients with Bernard-Soulier syndrome, congenitally deficient in GPI, both aggregated and released 14C-serotonin normally when exposed to latex particles coated with IgG. Levels of GPI were decreased experimentally in normal platelets by treating them with chymotrypsin. Platelets treated in this manner did not aggregate or release [14C]serotonin in response to ristocetin-VIII:vWf. They did, however, both aggregate and release when incubated with heat-aggregated IgG, antigen-antibody complexes or latex particles coated with IgG. Thus the presence of GPI is not a prerequisite for platelet stimulation via the Fc receptor. PMID:6231945

  15. Determination of KA values by controlled receptor expression in Xenopus oocytes.

    PubMed Central

    Murakoshi, H.; Nunoki, K.; Ishii, K.; Taira, N.

    1995-01-01

    1. In the present study we estimated the KA value of endothelin-1 (ET-1) for ETA-receptors by a new method in which the level of expression of ETA-receptors in Xenopus oocytes was altered in a controlled way. 2. Kvl.2 (a delayed rectifier type K channel) c RNA at the fixed concentration of 0.2 micro g micro l(-1) was mixed with ETA-receptor cRNA at various concentration ratios (10(-3)-3). Oocytes were examined 2-4 days after the injection of the cRNA mixtures. 3. In these oocytes, ET-1 suppressed the amplitude of Kvl.2 current in a dose-dependent manner in the range of 0.1-100 nM; the maximum inhibition produced by ET-1 was larger and the EC50 value for the inhibition by ET-1 was smaller as the mixture ratio was increased. Double-reciprocal plots of equiactive concentrations of ET-1 in 1/1- and 1/30-injected oocytes yielded a KA for ET-1 of 7.4 nM. The number of ETA-receptors in 1/30-injected oocytes was 13% of that in 1/1-injected oocytes, whereas the inhibition of the current in 1/30-injected oocytes was about 60% of that in 1/1-injected oocytes. This suggests the presence of spare receptors of ETA in the latter. 4. A saturation binding experiment estimated a KD value of 0.1 nM for ET-1 at ETA-receptors and the number of ETA-receptors in 1/30-injected oocytes was 23% of that in 1/1-injected ones. This value was not significantly different from that estimated by the above new method. However, there was a discrepancy between KA and KD, which could be due to factors unique to the expression system employed in the present study. PMID:8640346

  16. Receptors for B cell stimulatory factor 2. Quantitation, specificity, distribution, and regulation of their expression

    SciTech Connect

    Taga, T.; Kawanishi, Y.; Hardy, R.R.; Hirano, T.; Kishimoto, T.

    1987-10-01

    B cell stimulatory factor 2 receptors (BSF-2-R) were studied using radioiodinated recombinant BSF-2 with a specific activity of 6.16 X 10(13) cpm/g. Kinetic studies showed that binding of /sup 125/I-BSF-2 to CESS cells reached maximum level within 150 min at 0 degrees C. There was a single class of receptors with high affinity (Kd 3.4 X 10(-10) M) on CESS, and the number of receptors was 2700 per cell. Binding of /sup 125/I-BSF-2 to CESS was competitively inhibited by unlabeled BSF-2 but not by IL-1, IL-2, IFN-beta, IFN-gamma, and G-CSF, indicating the presence of the receptors specific for BSF-2. EBV-transformed B lymphoblastoid cell lines (CESS, SKW6-CL4, LCL13, and LCL14) expressed BSF-2-R, whereas Burkitt's lines did not. EBV or EBNA2 did not induce the expression of the receptors on Burkitt's cells. The plasma cell lines (ARH-77 and U266) expressed BSF-2-R, fitting the function of BSF-2 as plasma cell growth factor. Several other cell lines, the histiocytic line U937, the promyelocytic line HL60, the astrocytoma line U373 and the glioblastoma line SK-MG-4, in which BSF-2 was inducible with IL-1 or TPA, displayed BSF-2-R with Kd in the range of 1.3-6.4 X 10(-10) M, suggesting the autocrine mechanism in BSF-2 function. The four T cell lines (CEM, HSB, Jurkat, and OM 1) did not express a detectable number of receptors, but normal resting T cells expressed 100-1000 receptors per cell. BSF-2-R were not present on normal resting B cells but expressed on activated B cells with a Kd of 3.6-5.0 X 10(-10) M, fitting the function of BSF-2, which acts on B cells at the final maturation stage to induce immunoglobulin production.

  17. Metformin reduces endothelial cell expression of both the receptor for advanced glycation end products and lectin-like oxidized receptor 1.

    PubMed

    Ouslimani, Nadjat; Mahrouf, Meriem; Peynet, Jacqueline; Bonnefont-Rousselot, Dominique; Cosson, Claudine; Legrand, Alain; Beaudeux, Jean-Louis

    2007-03-01

    Beyond its antihyperglycemic action, the antidiabetic oral drug metformin possesses antioxidant properties that may contribute to improve the cardiovascular deleterious effects of the diabetic disease. We explored whether metformin could modulate the redox-sensible expression of receptor for advanced glycation end products (RAGE) and lectin-like oxidized receptor 1 (LOX-1), 2 endothelial membrane receptors involved in the arterial endothelial dysfunction observed in diabetes. Bovine aortic endothelial cells, either unstimulated or activated by high levels of glucose (30 mmol/L) or advanced glycation end products, were incubated for 72 hours with metformin at therapeutically relevant concentrations (10(-5) to 5 x 10(-4) mol/L). The expressions of RAGE and LOX-1 were evaluated on cell extracts by Western blot analysis. Metformin was shown to reduce, in dose-dependent manner, such expression of the 2 receptors, both in stimulated (by either glucose or advanced glycation end products) and in unstimulated cells. The effect of metformin was associated with a decrease in intracellular reactive oxygen species as assessed using the 2',7'-dichlorodihydrofluorescein diacetate fluoroprobe. Taken together, our results suggest that the intracellular antioxidant properties of metformin may result in the inhibition of cell expression of both RAGE and LOX-1, possibly through a modulation of redox-sensible nuclear factors such as nuclear factor kappaB, that were shown to be involved in such receptor cell expression. PMID:17292717

  18. The generation of knock-in mice expressing fluorescently tagged galanin receptors 1 and 2

    PubMed Central

    Kerr, Niall; Holmes, Fiona E.; Hobson, Sally-Ann; Vanderplank, Penny; Leard, Alan; Balthasar, Nina; Wynick, David

    2015-01-01

    The neuropeptide galanin has diverse roles in the central and peripheral nervous systems, by activating the G protein-coupled receptors Gal1, Gal2 and the less studied Gal3 (GalR1–3 gene products). There is a wealth of data on expression of Gal1–3 at the mRNA level, but not at the protein level due to the lack of specificity of currently available antibodies. Here we report the generation of knock-in mice expressing Gal1 or Gal2 receptor fluorescently tagged at the C-terminus with, respectively, mCherry or hrGFP (humanized Renilla green fluorescent protein). In dorsal root ganglia (DRG) neurons expressing the highest levels of Gal1-mCherry, localization to the somatic cell membrane was detected by live-cell fluorescence and immunohistochemistry, and that fluorescence decreased upon addition of galanin. In spinal cord, abundant Gal1-mCherry immunoreactive processes were detected in the superficial layers of the dorsal horn, and highly expressing intrinsic neurons of the lamina III/IV border showed both somatic cell membrane localization and outward transport of receptor from the cell body, detected as puncta within cell processes. In brain, high levels of Gal1-mCherry immunofluorescence were detected within thalamus, hypothalamus and amygdala, with a high density of nerve endings in the external zone of the median eminence, and regions with lesser immunoreactivity included the dorsal raphe nucleus. Gal2-hrGFP mRNA was detected in DRG, but live-cell fluorescence was at the limits of detection, drawing attention to both the much lower mRNA expression than to Gal1 in mice and the previously unrecognized potential for translational control by upstream open reading frames (uORFs). PMID:26292267

  19. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts

    PubMed Central

    Schütz, Marco; Schöppe, Jendrik; Sedlák, Erik; Hillenbrand, Matthias; Nagy-Davidescu, Gabriela; Ehrenmann, Janosch; Klenk, Christoph; Egloff, Pascal; Kummer, Lutz; Plückthun, Andreas

    2016-01-01

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation. PMID:26911446

  20. Directed evolution of G protein-coupled receptors in yeast for higher functional production in eukaryotic expression hosts.

    PubMed

    Schütz, Marco; Schöppe, Jendrik; Sedlák, Erik; Hillenbrand, Matthias; Nagy-Davidescu, Gabriela; Ehrenmann, Janosch; Klenk, Christoph; Egloff, Pascal; Kummer, Lutz; Plückthun, Andreas

    2016-01-01

    Despite recent successes, many G protein-coupled receptors (GPCRs) remained refractory to detailed molecular studies due to insufficient production yields, even in the most sophisticated eukaryotic expression systems. Here we introduce a robust method employing directed evolution of GPCRs in yeast that allows fast and efficient generation of receptor variants which show strongly increased functional production levels in eukaryotic expression hosts. Shown by evolving three different receptors in this study, the method is widely applicable, even for GPCRs which are very difficult to express. The evolved variants showed up to a 26-fold increase of functional production in insect cells compared to the wild-type receptors. Next to the increased production, the obtained variants exhibited improved biophysical properties, while functional properties remained largely unaffected. Thus, the presented method broadens the portfolio of GPCRs accessible for detailed investigations. Interestingly, the functional production of GPCRs in yeast can be further increased by induced host adaptation. PMID:26911446

  1. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  2. Murine Hematopoietic Stem cells and Progenitors Express Adrenergic Receptors

    PubMed Central

    Muthu, Kuzhali; Iyer, Sivaraman; He, L-K.; Szilagyi, Andrea; Gamelli, Richard L; Shankar, Ravi; Jones, Stephen B

    2007-01-01

    Association between the nervous and immune system is well documented. Immune cells originate within the bone marrow that is innervated. Thermal injury induces adrenergic stimulation, augments monocytopoiesis and alters the β-adrenergic receptor (AR) profile of bone marrow monocyte committed progenitors. This provides an impetus to study AR expression in hematopoietic progenitors along myeloid lineage. Using FACS analysis and confocal microscopy, we report the expression of α1-, α2- and β2- AR in enriched populations of ER-MP20+ and ER-MP12+ myeloid progenitors, CD117+ and CD34+ multi-potential progenitors and more importantly pluripotent stem cells suggesting a plausible role for catecholamine in hematopoietic development. PMID:17428548

  3. Epithelial P2X purinergic receptor channel expression and function

    PubMed Central

    Taylor, Amanda L.; Schwiebert, Lisa M.; Smith, Jeffrey J.; King, Chris; Jones, Julie R.; Sorscher, Eric J.; Schwiebert, Erik M.

    1999-01-01

    P2X purinergic receptor (P2XR) channels bind ATP and mediate Ca2+ influx — 2 signals that stimulate secretory Cl– transport across epithelia. We tested the hypotheses that P2XR channels are expressed by epithelia and that P2XRs transduce extracellular ATP signals into stimulation of Cl– transport across epithelia. Electrophysiological data and mRNA analysis of human and mouse pulmonary epithelia and other epithelial cells indicate that multiple P2XRs are broadly expressed in these tissues and that they are active on both apical and basolateral surfaces. Because P2X-selective agonists bind multiple P2XR subtypes, and because P2X agonists stimulate Cl– transport across nasal mucosa of cystic fibrosis (CF) patients as well as across non-CF nasal mucosa, P2XRs may provide novel targets for extracellular nucleotide therapy of CF. PMID:10510328

  4. Ionotropic glutamate receptor expression in human white matter.

    PubMed

    Christensen, Pia Crone; Samadi-Bahrami, Zahra; Pavlov, Vlady; Stys, Peter K; Moore, G R Wayne

    2016-09-01

    Glutamate is the key excitatory neurotransmitter of the central nervous system (CNS). Its role in human grey matter transmission is well understood, but this is less clear in white matter (WM). Ionotropic glutamate receptors (iGluR) are found on both neuronal cell bodies and glia as well as on myelinated axons in rodents, and rodent WM tissue is capable of glutamate release. Thus, rodent WM expresses many of the components of the traditional grey matter neuron-to-neuron synapse, but to date this has not been shown for human WM. We demonstrate the presence of iGluRs in human WM by immunofluorescence employing high-resolution spectral confocal imaging. We found that the obligatory N-methyl-d-aspartic acid (NMDA) receptor subunit GluN1 and the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor subunit GluA4 co-localized with myelin, oligodendroglial cell bodies and processes. Additionally, GluA4 colocalized with axons, often in distinct clusters. These findings may explain why human WM is vulnerable to excitotoxic events following acute insults such as stroke and traumatic brain injury and in more chronic inflammatory conditions such as multiple sclerosis (MS). Further exploration of human WM glutamate signalling could pave the way for developing future therapies modulating the glutamate-mediated damage in these and other CNS disorders. PMID:27443784

  5. Perfluorooctane sulfonate (PFOS) affects hormone receptor activity, steroidogenesis, and expression of endocrine-related genes in vitro and in vivo.

    PubMed

    Du, Guizhen; Hu, Jialei; Huang, Hongyu; Qin, Yufeng; Han, Xiumei; Wu, Di; Song, Ling; Xia, Yankai; Wang, Xinru

    2013-02-01

    Perfluorooctane sulfonate (PFOS) is a widespread and persistent chemical in the environment. We investigated the endocrine-disrupting effects of PFOS using a combination of in vitro and in vivo assays. Reporter gene assays were used to detect receptor-mediated (anti-)estrogenic, (anti-)androgenic, and (anti-)thyroid hormone activities. The effect of PFOS on steroidogenesis was assessed both at hormone levels in the supernatant and at expression levels of hormone-induced genes in the H295R cell. A zebrafish-based short-term screening method was developed to detect the effect of PFOS on endocrine function in vivo. The results indicate that PFOS can act as an estrogen receptor agonist and thyroid hormone receptor antagonist. Exposure to PFOS decreased supernatant testosterone (T), increased estradiol (E2) concentrations in H295R cell medium and altered the expression of several genes involved in steroidogenesis. In addition, PFOS increased early thyroid development gene (hhex and pax8) expression in a concentration-dependent manner, decreased steroidogenic enzyme gene (CYP17, CYP19a, CYP19b) expression, and changed the expression pattern of estrogen receptor production genes (esr1, esr2b) after 500 µg/L PFOS treatment in zebrafish embryos. These results indicate that PFOS has the ability to act as an endocrine disruptor both in vitro and in vivo by disrupting the function of nuclear hormone receptors, interfering with steroidogenesis, and altering the expression of endocrine-related genes in zebrafish embryo. PMID:23074026

  6. Differential effects of diazepam treatment and withdrawal on recombinant GABAA receptor expression and functional coupling.

    PubMed

    Svob Strac, Dubravka; Vlainić, Josipa; Jazvinsćak Jembrek, Maja; Pericić, Danka

    2008-12-30

    Prolonged exposure to benzodiazepines, drugs known to produce tolerance and dependence and also to be abused, leads to adaptive changes in GABA(A) receptors. To further explore the mechanisms responsible for these phenomena, we studied the effects of prolonged diazepam treatment on the recombinant alpha(1)beta(2)gamma(2S) GABA(A) receptors, stably expressed in human embryonic kidney (HEK) 293 cells. The results demonstrating that long-term (48 and 72 h) exposure of cells to a high concentration of diazepam (50 microM) enhanced the maximum number (B(max)) of [(3)H]flunitrazepam, [(3)H]muscimol and [(3)H]t-butylbicycloorthobenzoate ([(3)H]TBOB) binding sites, without changing their affinity (K(d)), suggested the up-regulation of GABA(A) receptors. As demonstrated by cell counting and WST-1 proliferation assay, the observed increase in receptor expression was not a consequence of stimulated growth of cells exposed to diazepam. Semi-quantitative RT-PCR and Western blot analysis, showing elevated levels of alpha(1) subunit mRNA as well as beta(2) and gamma(2) subunit proteins, respectively, suggested that prolonged high dose diazepam treatment induced de novo receptor synthesis by acting at both transcriptional and translational levels. The finding that the number of GABA(A) receptor binding sites returned to control value 24 h following diazepam withdrawal, makes this process less likely to account for the development of benzodiazepine tolerance and dependence. On the other hand, the results demonstrating that observed functional uncoupling between GABA and benzodiazepine binding sites persisted after the termination of diazepam treatment supported the hypothesis of its possible role in these phenomena. PMID:18955034

  7. Toll-like receptor 2 ligands regulate monocyte Fcγ receptor expression and function.

    PubMed

    Shah, Prexy; Fatehchand, Kavin; Patel, Hemal; Fang, Huiqing; Justiniano, Steven E; Mo, Xiaokui; Jarjoura, David; Tridandapani, Susheela; Butchar, Jonathan P

    2013-04-26

    Fcγ receptor (FcγR) clustering on monocytes/macrophages results in phagocytosis and inflammatory cytokine production, which serve to eliminate antibody-opsonized targets and activate neighboring immune cells. Toll-like receptor 2 (TLR2), which recognizes a range of both bacterial and fungal components, elicits strong proinflammatory responses in these cells when stimulated by ligands, either natural or synthetic. Thus, we explored the possibility that TLR2 agonists could strengthen FcγR activity within the context of antibody therapy. Human peripheral blood monocytes treated with the TLR2 agonist Pam2CSK4 showed significantly enhanced FcγR-mediated cytokine production as well as phagocytic ability. An examination of the molecular mechanism behind this enhancement revealed increased expression of both FcγRIIa and the common γ subunit following Pam2CSK4 treatment. Interestingly however, expression of the inhibitory receptor FcγRIIb was also modestly increased. Further investigation revealed that Pam2CSK4 also dramatically decreased the expression of SHIP, the major mediator of FcγRIIb inhibitory activity. Using a murine Her2/neu solid tumor model of antibody therapy, we found that Pam2CSK4 significantly enhanced the ability of anti-Her2 antibody to reduce the rate of tumor growth. To verify that the FcγR enhancement was not unique to the diacylated Pam2CSK4, we also tested Pam3CSK4, a related triacylated TLR2 agonist. Results showed significant enhancement in FcγR function and expression. Taken together, these findings indicate that TLR2 activation can positively modulate FcγR and suggest that TLR2 agonists should be considered for testing as adjuvants for antitumor antibody therapy. PMID:23504312

  8. Poly(ADP-ribose) Polymerase Inhibitors Sensitize Cancer Cells to Death Receptor-mediated Apoptosis by Enhancing Death Receptor Expression*

    PubMed Central

    Meng, X. Wei; Koh, Brian D.; Zhang, Jin-San; Flatten, Karen S.; Schneider, Paula A.; Billadeau, Daniel D.; Hess, Allan D.; Smith, B. Douglas; Karp, Judith E.; Kaufmann, Scott H.

    2014-01-01

    Recombinant human tumor necrosis factor-α-related apoptosis inducing ligand (TRAIL), agonistic monoclonal antibodies to TRAIL receptors, and small molecule TRAIL receptor agonists are in various stages of preclinical and early phase clinical testing as potential anticancer drugs. Accordingly, there is substantial interest in understanding factors that affect sensitivity to these agents. In the present study we observed that the poly(ADP-ribose) polymerase (PARP) inhibitors olaparib and veliparib sensitize the myeloid leukemia cell lines ML-1 and K562, the ovarian cancer line PEO1, non-small cell lung cancer line A549, and a majority of clinical AML isolates, but not normal marrow, to TRAIL. Further analysis demonstrated that PARP inhibitor treatment results in activation of the FAS and TNFRSF10B (death receptor 5 (DR5)) promoters, increased Fas and DR5 mRNA, and elevated cell surface expression of these receptors in sensitized cells. Chromatin immunoprecipitation demonstrated enhanced binding of the transcription factor Sp1 to the TNFRSF10B promoter in the presence of PARP inhibitor. Knockdown of PARP1 or PARP2 (but not PARP3 and PARP4) not only increased expression of Fas and DR5 at the mRNA and protein level, but also recapitulated the sensitizing effects of the PARP inhibition. Conversely, Sp1 knockdown diminished the PARP inhibitor effects. In view of the fact that TRAIL is part of the armamentarium of natural killer cells, these observations identify a new facet of PARP inhibitor action while simultaneously providing the mechanistic underpinnings of a novel therapeutic combination that warrants further investigation. PMID:24895135

  9. The expression of several reproductive hormone receptors can be modified by perfluorooctane sulfonate (PFOS) in adult male rats.

    PubMed

    López-Doval, S; Salgado, R; Lafuente, A

    2016-07-01

    This study was undertaken to evaluate the possible role of several reproductive hormone receptors on the disruption of the hypothalamic-pituitary-testis (HPT) axis activity induced by perfluorooctane sulfonate (PFOS). The studied receptors are the gonadotropin-releasing hormone receptor (GnRHr), luteinizing hormone receptor (LHr), follicle-stimulating hormone receptor (FSHr), and the androgen receptor (Ar). Adult male rats were orally treated with 1.0; 3.0 and 6.0 mg of PFOS kg(-1) d(-1) for 28 days. In general terms, PFOS can modify the relative gene and protein expressions of these receptors in several tissues of the reproductive axis. At the testicular level, apart from the expected inhibition of both gene and protein expressions of FSHr and Ar, PFOS also stimulates the GnRHr protein and the LHr gene expression. The receptors of the main hormones involved in the HPT axis may have an important role in the disruption exerted by PFOS on this axis. PMID:27151425

  10. Rapamycin down-regulates LDL-receptor expression independently of SREBP-2

    SciTech Connect

    Sharpe, Laura J.; Brown, Andrew J.

    2008-09-05

    As a key regulator of cholesterol homeostasis, sterol-regulatory element binding protein-2 (SREBP-2) up-regulates expression of genes involved in cholesterol synthesis (e.g., 3-hydroxy-3-methyl-glutaryl-coenzyme A (HMG-CoA) Reductase) and uptake (the low density lipoprotein (LDL)-receptor). Previously, we showed that Akt, a critical kinase in cell growth and proliferation, contributes to SREBP-2 activation. However, the specific Akt target involved is unknown. A potential candidate is the mammalian target of rapamycin, mTOR. Rapamycin can cause hyperlipidaemia clinically, and we hypothesised that this may be mediated via an effect of mTOR on SREBP-2. Herein, we found that SREBP-2 activation and HMG-CoA Reductase gene expression were unaffected by rapamycin treatment. However, LDL-receptor gene expression was decreased by rapamycin, suggesting that this may contribute to the hyperlipidaemia observed in rapamycin-treated patients. Rapamycin did not affect mRNA stability, so the decrease in LDL-receptor gene expression is likely to be occurring at the transcriptional level, although independently of SREBP-2.

  11. The flavonoid apigenin suppresses vitamin D receptor expression and vitamin D responsiveness in normal human keratinocytes.

    PubMed

    Segaert, S; Courtois, S; Garmyn, M; Degreef, H; Bouillon, R

    2000-02-01

    Apigenin, a flavonoid with chemopreventive properties, induces cellular growth arrest, with concomitant inhibition of intracellular signaling cascades and decreased proto-oncogene expression. We report that apigenin potently inhibited vitamin D receptor (VDR) mRNA and protein expression in human keratinocytes without changes in VDR mRNA half-life. Concurrently, downregulation of retinoid X receptor alpha, a dramatic loss of c-myc mRNA, and upregulation of p21(WAF1) took place. Furthermore, a nearly complete suppression of vitamin D responsiveness was observed as estimated by induction of 24-hydroxylase mRNA. The apigenin effect on VDR expression was shared by some other (quercetine and fisetine) but not all tested flavonoids. Interestingly, the apigenin-mediated VDR suppression was counteracted by the NFkappaB inhibitors sodium salicylate and caffeic acid phenethyl ester. The presented results propose suppression of nuclear receptor levels as a novel mechanism whereby flavonoids exert their pleiotropic effects. This study may also contribute to the understanding of the regulation of VDR expression in epidermal keratinocytes. PMID:10652242

  12. Interleukin-18 Increases TLR4 and Mannose Receptor Expression and Modulates Cytokine Production in Human Monocytes

    PubMed Central

    Dias-Melicio, Luciane Alarcão; Fernandes, Reginaldo Keller; Rodrigues, Daniela Ramos; Golim, Marjorie Assis; Soares, Angela Maria Victoriano Campos

    2015-01-01

    Interleukin-18 is a proinflammatory cytokine belonging to the interleukin-1 family of cytokines. This cytokine exerts many unique biological and immunological effects. To explore the role of IL-18 in inflammatory innate immune responses, we investigated its impact on expression of two toll-like receptors (TLR2 and TLR4) and mannose receptor (MR) by human peripheral blood monocytes and its effect on TNF-α, IL-12, IL-15, and IL-10 production. Monocytes from healthy donors were stimulated or not with IL-18 for 18 h, and then the TLR2, TLR4, and MR expression and intracellular TNF-α, IL-12, and IL-10 production were assessed by flow cytometry and the levels of TNF-α, IL-12, IL-15, and IL-10 in culture supernatants were measured by ELISA. IL-18 treatment was able to increase TLR4 and MR expression by monocytes. The production of TNF-α and IL-10 was also increased by cytokine treatment. However, IL-18 was unable to induce neither IL-12 nor IL-15 production by these cells. Taken together, these results show an important role of IL-18 on the early phase of inflammatory response by promoting the expression of some pattern recognition receptors (PRRs) that are important during the microbe recognition phase and by inducing some important cytokines such as TNF-α and IL-10. PMID:25873755

  13. Neonatal 6-hydroxydopamine lesions lead to opposing changes in the levels of dopamine receptors and their messenger RNAs.

    PubMed

    Frohna, P A; Neal-Beliveau, B S; Joyce, J N

    1995-09-01

    Previous studies have established that selective damage to the early-developing components of the mesostriatal dopamine system produces profound changes in dopamine D1 receptor-mediated behaviors, while decreasing D1 receptor density. In order to better understand the effects of early intrastriatal 6-hydroxydopamine lesions, we studied the ontogenetic expression (postnatal days 7, 14, 35 and 90) of D1 and D2 receptors, and their corresponding messenger RNAs, in rats that had received intrastriatal 6-hydroxydopamine or vehicle lesions on postnatal day 1. Using receptor autoradiography, significant (P < 0.05) decreases in [3H]SCH 23390 binding to D1 receptors in the rostral and caudal dorsomedial and ventromedial caudate of 6-hydroxydopamine-lesioned animals were evident by postnatal day 7, and remained depressed at all future time points. A significant decrease in D1 receptor concentration occurred in the dorsolateral caudate at later time points (postnatal days 35 and 90). [3H]Spiperone binding to D2 receptor sites was unchanged throughout the entire study. In situ hybridization for D1 and D2 messenger RNA expression showed contrasting results. 6-Hydroxydopamine induced significant decreases of D1 messenger RNA levels in the dorsolateral and dorsomedial caudate by postnatal day 7. By postnatal day 14, messenger RNA expression was significantly elevated in the dorsomedial and ventromedial caudate of the 6-hydroxydopamine group, and remained elevated thereafter. D1 messenger RNA levels became elevated in the lateral caudate at later time points (postnatal days 35 and 90). The opposing changes in D1 receptor concentrations and the messenger RNA encoding the protein did not occur as a consequence of increased transport of D1 receptors to striatonigral terminals. D2 messenger RNA levels in the dorsal caudate were significantly decreased on postnatal day 7, and became higher than controls at postnatal day 14, but were unchanged from controls at later time points

  14. Molecular cloning, characterization, and expression profiles of androgen receptors in spotted scat (Scatophagus argus).

    PubMed

    Chen, H P; Deng, S P; Dai, M L; Zhu, C H; Li, G L

    2016-01-01

    Androgen plays critical roles in vertebrate reproductive systems via androgen receptors (ARs). In the present study, the full-length spotted scat (Scatophagus argus) androgen receptor (sAR) cDNA sequence was cloned from testis. The sAR cDNA measured 2448 bp in length with an open-reading frame of 2289 bp, encoding 763 amino acids. Amino acid alignment analyses showed that the sARs exhibited highly evolutionary conserved functional domains. Phylogenetically, the sARs clustered within the ARβ common vertebrate group. Real-time polymerase chain reaction (RT-PCR) revealed that sAR expression varied in level and distribution throughout the tissues of both females and males. sAR expression was detected during testicular development by quantitative RT-PCR. The results showed that the highest transcription of sARs was observed in the mid-testicular stage, and remained at a high expression level until the late-testicular stage. In addition, the effects of 17α-methyltestosterone (MT) and estrogen (E2) on the expression of sARs in ovaries were determined using quantitative RT-PCR. sAR expression increased at 12 and 24 h post-MT treatment and decreased with E2 treatment. The present study provides preliminary evidence indicating gonadal plasticity of spotted scat under exogenous steroidal hormone treatments. It also provides a theoretical basis for sex reversal and production of artificial pseudo-males for female monosex breeding. PMID:27173207

  15. Increased endometrial expression of CC-chemokine receptor-1 in women with adenomyosis.

    PubMed

    Xu, Hong; Yang, Yanfeng; Zhou, Caiyun; Huang, Xiufeng; Lin, Jun; Zhang, Xinmei

    2014-09-01

    Abnormal endometrial expression of CC-chemokine receptor-1 (CCR1) may play a role in the pathogenesis of endometriosis. Adenomyosis, also called endometriosis interna, occurs when the endometrium invades the myometrium. The objective of this study was to determine CCR1 expression in endometrium in women with adenomyosis as compared to women without adenomyosis. We evaluated endometrial mRNA and protein expression in women with and without adenomyosis using quantitative polymerase chain reaction (PCR), immunohistochemical staining and western blot analysis, respectively. We detected CCR1-immunoreactive expression in endometrium in all women with and without adenomyosis. CCR1-immunoreactive staining in endometrial cells was significantly higher in women with adenomyosis (4.89±1.06) compared to those without adenomyosis (2.21±1.16, P<0.001). Women with adenomyosis had higher levels of CCR1 mRNA in endometrium compared to women without adenomyosis (P<0.05). CCR1 protein levels in endometrium were significantly higher in women with adenomyosis (1.66±0.79) compared to women without adenomyosis (0.56±0.13, P<0.001), and positively correlated with the severity of dysmenorrhea (r=0.87, P<0.001). These results suggest that increased CC-chemokine receptor expression may play a role in the pathogenesis of adenomyosis. PMID:24599574

  16. Canonical transient receptor potential channels expression is elevated in a porcine model of metabolic syndrome.

    PubMed

    Hu, Guoqing; Oboukhova, Elena A; Kumar, Sanjay; Sturek, Michael; Obukhov, Alexander G

    2009-05-01

    Plasma epinephrine and heart rate are elevated in metabolic syndrome, suggesting enhanced catecholamine secretion from the adrenal medulla. Canonical transient receptor potential (TRPC) channels are implicated in mediating hormone-induced Ca(2+) influx and catecholamine secretion in adrenomedullary chromaffin cells. We studied the pattern of TRPC expression in the pig adrenal medulla and investigated whether adrenal TRPC expression is altered in prediabetic metabolic syndrome Ossabaw miniature pigs. We used a combination of molecular biological, biochemical, and fluorescence imaging techniques. We determined the sequence of pig TRPC1 and TRPC3-7 channels. We found that the pig adrenal medulla expressed predominantly TRPC1, TRPC5, and TRPC6 transcripts. The expression level of these TRPCs was significantly elevated in the adrenal medulla from pigs with metabolic syndrome. Interestingly, aldosterone, which is endogenously secreted in the adjacent adrenal cortex, increased TRPC1, TRPC5, and TRPC6 expression in adrenal chromaffin cells isolated from metabolic syndrome but not control pigs. Spironolactone, a blocker of mineralocorticoid receptors, inhibited the aldosterone effect. Dexamethasone also increased TRPC5 expression in metabolic syndrome chromaffin cells. The amplitude of hormone-induced divalent cation influx correlated with the level of TRPC expression in adrenal chromaffin cells. Orai1/Stim1 protein expression was not significantly altered in the metabolic syndrome adrenal medulla when compared with the control. We propose that in metabolic syndrome, abnormally elevated adrenal TRPC expression may underlie increased plasma epinephrine and heart rate. The excess of plasma catecholamines and increased heart rate are risk factors for cardiovascular disease. Thus, TRPCs are potential therapeutic targets in the fight against cardiovascular disease. PMID:19221052

  17. Cloning and expression of an A1 adenosine receptor from rat brain

    SciTech Connect

    Mahan, L.C.; McVittie, L.D.; Smyk-Randall, E.M.; Nakata, H.; Monsma, F.J. Jr.; Gerfen, C.R.; Sibley, D.R. )

    1991-07-01

    The authors have used the polymerase chain reaction technique to selectively amplify guanine nucleotide-binding regulatory protein (G protein)-coupled receptor cDNA sequences from rat striatal mRNA, using sets of highly degenerate primers derived from transmembrane sequences of previously cloned G protein-coupled receptors. A novel cDNA fragment was identified, which exhibits considerable homology to various members of the G protein-coupled receptor family. This fragment was used to isolate a full-length cDNA from a rat striatal library. A 2.2-kilobase clone was obtained that encodes a protein of 326 amino acids with seven transmembrane domains, as predicted by hydropathy analysis. Stably transfected mouse A9-L cells and Chinese hamster ovary cells that expressed mRNA for this clone were screened with putative receptor ligands. Saturable and specific binding sites for the A1 adenosine antagonist (3H)-1,3-dipropyl-8-cyclopentylxanthine were identified on membranes from transfected cells. The rank order of potency and affinities of various adenosine agonist and antagonist ligands confirmed the identity of this cDNA clone as an A1 adenosine receptor. The high affinity binding of A1 adenosine agonists was shown to be sensitive to the nonhydrolyzable GTP analog guanylyl-5{prime}-imidodiphosphate. In adenylyl cyclase assays, adenosine agonists inhibited forskolin-stimulated cAMP production by greater than 50%, in a pharmacologically specific fashion. Northern blot and in situ hybridization analyses of receptor mRNA in brain tissues revealed two transcripts of 5.6 and 3.1 kilobases, both of which were abundant in cortex, cerebellum, hippocampus, and thalamus, with lower levels in olfactory bulb, striatum, mesencephalon, and retina. These regional distribution data are in good agreement with previous receptor autoradiographic studies involving the A1 adenosine receptor.

  18. Increased expression of estrogen-related receptor β during adaptation of adult cardiomyocytes to sustained hypoxia

    PubMed Central

    Cunningham, Kathryn F; Beeson, Gyda C; Beeson, Craig C; McDermott, Paul J

    2016-01-01

    Estrogen-related Receptors (ERR) are members of the steroid hormone receptor superfamily of transcription factors that regulate expression of genes required for energy metabolism including mitochondrial biogenesis, fatty acid oxidation and oxidative phosphorylation. While ERRα and EPPγ isoforms are known to share a wide array of target genes in the adult myocardium, the function of ERRβ has not been characterized in cardiomyocytes. The purpose of this study was to determine the role of ERRβ in regulating energy metabolism in adult cardiomyocytes in primary culture. Adult feline cardiomyocytes were electrically stimulated to contract in either hypoxia (0.5% O2) or normoxia (21% O2). As compared to baseline values measured in normoxia, ERRβ mRNA levels increased significantly after 8 hours of hypoxia and remained elevated over 24 h. Conversely, ERRβ mRNA decreased to normoxic levels after 4 hours of reoxygenation. Hypoxia increased expression of the α and β isoforms of Peroxisome Proliferator-Activated Receptor γ Coactivator-1 (PGC-1) mRNA by 6-fold and 3-fold, respectively. Knockdown of ERRβ expression via adenoviral-mediated delivery of ERRβ shRNA blocked hypoxia-induced increases in PGC-1β mRNA, but not PGC-1α mRNA. Loss of ERRβ had no effect on mtDNA content as measured after 24 h of hypoxia. To determine whether loss of ERRβ affected mitochondrial function, oxygen consumption rates (OCR) were measured in contracting versus quiescent cardiomyocytes in normoxia. OCR was significantly lower in contracting cardiomyocytes expressing ERRβ shRNA than scrambled shRNA controls. Maximal OCR also was reduced by ERRβ knockdown. In conclusion: 1) hypoxia increases in ERRβ mRNA expression in contracting cardiomyocytes; 2) ERRβ is required for induction of the PGC-1β isoform in response to hypoxia; 3) ERRβ expression is required to sustain OCR in normoxic conditions. PMID:27335690

  19. Changes in estrogen receptor expression in the chick thymus during late embryonic development.

    PubMed

    Katayama, Masafumi; Fukuda, Tomokazu; Hatabu, Toshimitsu; Narabara, Kiyoaki; Abe, Asaki; Kondo, Yasuhiro

    2014-03-01

    In chickens, although estrogen receptors (ER) are reported to be associated with the immunological processes, detailed information about the differences in ER expression in the tissues related to the development of lymphocytes is not fully known, especially during the developmental stage. To learn more about this immunological relationship, we used semi-quantitative polymerase chain reaction method to detect the ER expression levels in the thymus tissues of chicks during the developmental stage. Furthermore, ER-expressing cells were detected by immunohistochemistry. The results of this study show that the expression level of ER increased on embryonic day 16 and decreased on day 20. Furthermore, ER expression was significantly higher in male than in female chickens at day 16. The increased expression on day 16 and decreased level on day 20 were also reproduced in the incidence of immunoreactive cells, although there was a 1-day delay in the elevated incidence of the cells. This study revealed the changes in ER expression and the incidence of ER-positive cells in the thymus of chickens during the developmental stage. PMID:24000785

  20. Peroxisome proliferator-activated receptor-alpha expression in rat liver during postnatal development.

    PubMed

    Panadero, M; Herrera, E; Bocos, C

    2000-08-01

    The expression of the peroxisome proliferator-activated receptor-alpha (PPARalpha) as well as of some related genes was studied in rat liver at different stages of development (from 19-day-old fetuses to 1 month-old rats). The level of PPARalpha mRNA appeared higher in neonates than in fetuses or 1 month-old rats. Whereas the pattern for phosphoenolpyruvate carboxykinase (PEPCK) mRNA level was similar to that of PPARalpha, the mRNA level of both acyl-CoA oxidase (ACO) and apolipoprotein CIII (apo CIII) showed diverse profiles. Western blotting analysis also revealed an increased level of PPARalpha protein in liver of suckling rats. Similarities of mRNA PEPCK and PPARalpha expression indicate a common control mechanism, where both nutritional and hormonal factors may be involved. PMID:11018288

  1. Nicotine induces fibrogenic changes in human liver via nicotinic acetylcholine receptors expressed on hepatic stellate cells

    SciTech Connect

    Soeda, Junpei; Morgan, Maelle; McKee, Chad; Mouralidarane, Angelina; Lin, ChingI; Roskams, Tania; Oben, Jude A.

    2012-01-06

    Highlights: Black-Right-Pointing-Pointer Cigarette smoke may induce liver fibrosis via nicotine receptors. Black-Right-Pointing-Pointer Nicotine induces proliferation of hepatic stellate cells (HSCs). Black-Right-Pointing-Pointer Nicotine activates hepatic fibrogenic pathways. Black-Right-Pointing-Pointer Nicotine receptor antagonists attenuate HSC proliferation. Black-Right-Pointing-Pointer Nicotinic receptor antagonists may have utility as novel anti-fibrotic agents. -- Abstract: Background and aims: Cigarette smoke (CS) may cause liver fibrosis but possible involved mechanisms are unclear. Among the many chemicals in CS is nicotine - which affects cells through nicotinic acetylcholine receptors (nAChR). We studied the effects of nicotine, and involved pathways, on human primary hepatic stellate cells (hHSCs), the principal fibrogenic cells in the liver. We then determined possible disease relevance by assaying nAChR in liver samples from human non-alcoholic steatohepatitis (NASH). Methods: hHSC were isolated from healthy human livers and nAChR expression analyzed - RT-PCR and Western blotting. Nicotine induction of hHSC proliferation, upregulation of collagen1-{alpha}2 and the pro-fibrogenic cytokine transforming growth factor beta 1 (TGF-{beta}1) was determined along with involved intracellular signaling pathways. nAChR mRNA expression was finally analyzed in whole liver biopsies obtained from patients diagnosed with non-alcoholic steatohepatitis (NASH). Results: hHSCs express muscle type ({alpha}1, {beta}1, delta and epsilon) and neuronal type ({alpha}3, {alpha}6, {alpha}7, {beta}2 and {beta}4) nAChR subunits at the mRNA level. Among these subunits, {alpha}3, {alpha}7, {beta}1 and {epsilon} were predominantly expressed as confirmed by Western blotting. Nicotine induced hHSC proliferation was attenuated by mecamylamine (p < 0.05). Additionally, collagen1-{alpha}2 and TGF-{beta}1 mRNA expression were significantly upregulated by nicotine and inhibited by

  2. Alteration in contractile G-protein coupled receptor expression by moist snus and nicotine in rat cerebral arteries

    SciTech Connect

    Sandhu, Hardip; Xu Cangbao; Edvinsson, Lars

    2011-04-15

    The cardiovascular risk for users of use of Swedish snus/American snuff (moist tobacco) has been debated for a long time. The present study was designed to examine the effects of water- or lipid-soluble (DMSO-soluble) snus and nicotine, the most important substance in tobacco, on the expression of vasocontractile G-protein coupled receptors (GPCR), such as endothelin ET{sub B}, serotonin 5-HT{sub 1B}, and thromboxane A{sub 2} TP receptors, in rat cerebral arteries. Studies show that these vasocontractile GPCR show alterations by lipid-soluble cigarette smoke particles via activation of mitogen-activated protein kinases (MAPK). However, the effects of moist tobacco on the expression of GPCR are less studied. Rat middle cerebral arteries were isolated and organ cultured in serum-free medium for 24 h in the presence of water-soluble snus (WSS), DMSO-soluble snus (DSS), or nicotine. The dose of snus and nicotine was kept at plasma level of snus users (25 ng nicotine/ml). A high dose (250 ng nicotine/ml) was also included due to the previous results showing alteration in the GPCR expression by nicotine at this concentration. Contractile responses to the ET{sub B} receptor agonist sarafotoxin 6c, 5-HT{sub 1B} receptor agonist 5-carboxamidotryptamine, and TP receptor agonist U46619 were investigated by a sensitive myograph. The expression of ET{sub B}, 5-HT{sub 1B}, and TP receptors was studied at mRNA and protein levels using quantitative real-time PCR and immunohistochemistry, respectively. Organ culture with WSS or DSS (25 ng nicotine/ml) lowered the 5-HT{sub 1B} receptor-mediated contraction. Furthermore, DSS shifted the TP receptor-mediated contraction curve left-wards with a stronger contraction. High dose of nicotine (250 ng nicotine/ml) increased the ET{sub B} receptor-mediated contraction. The combined 5-HT{sub 1B} and 5-HT{sub 2A} receptor-mediated contraction was increased, and both the 5-CT and TxA2 induced contractions were left-ward shifted by WSS, DSS, or

  3. A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

    PubMed Central

    McMahan, C J; Slack, J L; Mosley, B; Cosman, D; Lupton, S D; Brunton, L L; Grubin, C E; Wignall, J M; Jenkins, N A; Brannan, C I

    1991-01-01

    cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor. Images PMID:1833184

  4. Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection

    PubMed Central

    Yeaman, Grant R; Howell, Alexandra L; Weldon, Sally; Demian, Douglas J; Collins, Jane E; O'Connell, Denise M; Asin, Susana N; Wira, Charles R; Fanger, Michael W

    2003-01-01

    Human immunodeficiency virus-1 (HIV-1) is primarily a sexually transmitted disease. Identification of cell populations within the female reproductive tract that are initially infected, and the events involved in transmission of infection to other cells, remain to be established. In this report, we evaluated expression of HIV receptors and coreceptors on epithelial cells in the uterus and found they express several receptors critical for HIV infection including CD4, CXCR4, CCR5 and galactosylceramide (GalC). Moreover, expression of these receptors varied during the menstrual cycle. Expression of CD4 and CCR5 on uterine epithelial cells is high throughout the proliferative phase of the menstrual cycle when blood levels of oestradiol are high. In contrast, CXCR4 expression increased gradually throughout the proliferative phase. During the secretory phase of the cycle when both oestradiol and progesterone are elevated, CD4 and CCR5 expression decreased whereas CXCR4 expression remained elevated. Expression of GalC on endometrial glands is higher during the secretory phase than during the proliferative phase of the menstrual cycle. Because epithelial cells line the female reproductive tract and express HIV receptors and coreceptors, it is likely that they are one of the first cell types to become infected. The hormonal regulation of HIV receptor expression may affect a woman's susceptibility to HIV infection during her menstrual cycle. Moreover, selective coreceptor expression could account for the preferential transmission of R5-HIV-1 strains to women. In addition, these studies provide evidence that the uterus, and potentially the entire upper reproductive tract, are important sites for the initial events involved in HIV infection. PMID:12709027

  5. Human immunodeficiency virus receptor and coreceptor expression on human uterine epithelial cells: regulation of expression during the menstrual cycle and implications for human immunodeficiency virus infection.

    PubMed

    Yeaman, Grant R; Howell, Alexandra L; Weldon, Sally; Demian, Douglas J; Collins, Jane E; O'Connell, Denise M; Asin, Susana N; Wira, Charles R; Fanger, Michael W

    2003-05-01

    Human immunodeficiency virus-1 (HIV-1) is primarily a sexually transmitted disease. Identification of cell populations within the female reproductive tract that are initially infected, and the events involved in transmission of infection to other cells, remain to be established. In this report, we evaluated expression of HIV receptors and coreceptors on epithelial cells in the uterus and found they express several receptors critical for HIV infection including CD4, CXCR4, CCR5 and galactosylceramide (GalC). Moreover, expression of these receptors varied during the menstrual cycle. Expression of CD4 and CCR5 on uterine epithelial cells is high throughout the proliferative phase of the menstrual cycle when blood levels of oestradiol are high. In contrast, CXCR4 expression increased gradually throughout the proliferative phase. During the secretory phase of the cycle when both oestradiol and progesterone are elevated, CD4 and CCR5 expression decreased whereas CXCR4 expression remained elevated. Expression of GalC on endometrial glands is higher during the secretory phase than during the proliferative phase of the menstrual cycle. Because epithelial cells line the female reproductive tract and express HIV receptors and coreceptors, it is likely that they are one of the first cell types to become infected. The hormonal regulation of HIV receptor expression may affect a woman's susceptibility to HIV infection during her menstrual cycle. Moreover, selective coreceptor expression could account for the preferential transmission of R5-HIV-1 strains to women. In addition, these studies provide evidence that the uterus, and potentially the entire upper reproductive tract, are important sites for the initial events involved in HIV infection. PMID:12709027

  6. Differential developmental trajectories for CB1 cannabinoid receptor expression in limbic/associative and sensorimotor cortical areas

    PubMed Central

    Heng, Lijun; Beverley, Joel A.; Steiner, Heinz; Tseng, Kuei Y.

    2010-01-01

    Cannabis use during adolescence is associated with an increased risk for schizophrenia and other disorders. The neuronal basis is unclear, but prefrontal cortical mechanisms have been implicated. Here, we investigated developmental changes in the endocannabinoid system by assessing expression and function of the CB1 cannabinoid receptor in prefrontal and other cortical areas in juvenile (postnatal day 25, P25), adolescent (P40) and adult (P70) rats. Overall, the expression of CB1 receptors in the cortex is highest in juveniles and drops thereafter towards adult levels. However, CB1 receptor expression follows distinct developmental trajectories in different cortical areas. The most pronounced and progressive decrease in CB1 expression was observed in medial prefrontal and other limbic/associative regions. In contrast, major changes in sensorimotor cortices occurred only after P40. We also assessed electrophysiological measures of CB1 receptor function and found that CB1-dependent inhibition of synaptic transmission in the prefrontal cortex follows the same developmental trajectory as observed for receptor expression. Together, these findings indicate that CB1 receptor-mediated signaling decreases during development, but is differentially regulated in limbic/associative vs. sensorimotor systems. Therefore, cannabis use during adolescence likely differentially affects limbic/associative and sensorimotor cortical circuits. PMID:20687106

  7. Cannabinoid receptor 1-expressing neurons in the nucleus accumbens.

    PubMed

    Winters, Bradley D; Krüger, Juliane M; Huang, Xiaojie; Gallaher, Zachary R; Ishikawa, Masago; Czaja, Krzysztof; Krueger, James M; Huang, Yanhua H; Schlüter, Oliver M; Dong, Yan

    2012-10-01

    Endocannabinoid signaling critically regulates emotional and motivational states via activation of cannabinoid receptor 1 (CB1) in the brain. The nucleus accumbens (NAc) functions to gate emotional and motivational responses. Although expression of CB1 in the NAc is low, manipulation of CB1 signaling within the NAc triggers robust emotional/motivational alterations related to drug addiction and other psychiatric disorders, and these effects cannot be exclusively attributed to CB1 located at afferents to the NAc. Rather, CB1-expressing neurons in the NAc, although sparse, appear to be critical for emotional and motivational responses. However, the cellular properties of these neurons remain largely unknown. Here, we generated a knock-in mouse line in which CB1-expressing neurons expressed the fluorescent protein td-Tomato (tdT). Using these mice, we demonstrated that tdT-positive neurons within the NAc were exclusively fast-spiking interneurons (FSIs). These FSIs were electrically coupled with each other, and thus may help synchronize populations/ensembles of NAc neurons. CB1-expressing FSIs also form GABAergic synapses on adjacent medium spiny neurons (MSNs), providing feed-forward inhibition of NAc output. Furthermore, the membrane excitability of tdT-positive FSIs in the NAc was up-regulated after withdrawal from cocaine exposure, an effect that might increase FSI-to-MSN inhibition. Taken together with our previous findings that the membrane excitability of NAc MSNs is decreased during cocaine withdrawal, the present findings suggest that the basal functional output of the NAc is inhibited during cocaine withdrawal by multiple mechanisms. As such, CB1-expressing FSIs are targeted by cocaine exposure to influence the overall functional output of the NAc. PMID:23012412

  8. Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum

    SciTech Connect

    Voith, G.; Dingermann, T.

    1995-11-01

    We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I{gamma} promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I{gamma} promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays. 36 refs., 4 figs.

  9. Expression of the human muscarinic receptor gene m2 in Dictyostelium discoideum.

    PubMed

    Voith, G; Dingermann, T

    1995-11-01

    We have expressed a functional human muscarinic M2 receptor, under the control of the homologous discoidin I gamma promoter, in the cellular slime mold Dictyostelium discoideum. The use of a contact site A leader peptide ensured insertion of the newly synthesized receptor protein into the plasma membrane. Due to the characteristics of the discoidin I gamma promoter, the M2 receptor is expressed during late growth and early development. The heterologously expressed M2 receptors show binding characteristics similar to authentic receptors. Membranes as well as whole cells can be used in ligand binding assays. PMID:9636297

  10. Effect of stress hormones on the expression of fibrinogen-binding receptors in platelets.

    PubMed

    Lam, Nicole Y-L; Rainer, Timothy H; Ng, Margaret H-L; Leung, Yonna; Cocks, Robert A

    2002-12-01

    Acute coagulopathy is a common clinical complication after trauma, and contributes to posttraumatic multiple organ failure. The phenomenon may be due to the effect of stress hormones on platelet adhesion molecule expression after trauma. Catecholamine levels correlate with injury severity scores and changes of L-selectin expression on leucocytes, whilst adrenaline (ADR) (epinephrine) alone also activates platelets. This study thus investigates the effects of ADR and noradrenaline (NOR) (norepinephrine) on platelets, at doses similar to those found in the plasma of normal and trauma subjects. Blood was taken from 19 healthy subjects and placed in tubes containing sodium citrate. Anti-platelet-bound fibrinogen monoclonal antibody was used to identify the activated platelets while anti-CD41 was used to identify platelets with and without activation. Five increasing concentrations of ADR and NOR (1, 3, 5, 10, 30 nmol/l) as well as one negative control (0.9% normal saline) and one positive control (10 micromol/l adenosine diphosphate/ADP) were prepared for the stimulation. A whole blood protocol was used in order to minimize any activation artefacts, which might be created by centrifugation. The percentage of platelets expressing fibrinogen receptors increased significantly with ADR and NOR even at the lowest dose (1 nmol/l) and continued to increase in a dose-dependent manner. Although the effect of ADR was greater than NOR in stimulating platelets to express fibrinogen receptors, the average number of fibrinogen receptors on each platelet was constant. ADR and NOR activated platelets to express fibrinogen receptors at doses that are similar to those found in the plasma of trauma patients. PMID:12458065

  11. Allergic sensitization modifies the pulmonary expression of 5-hydroxytryptamine receptors in guinea pigs.

    PubMed

    Córdoba-Rodríguez, Guadalupe; Vargas, Mario H; Ruiz, Víctor; Carbajal, Verónica; Campos-Bedolla, Patricia; Mercadillo-Herrera, Paulina; Arreola-Ramírez, José Luis; Segura-Medina, Patricia

    2016-03-01

    There is mounting evidence that 5-hydroxytryptamine (5-HT) plays a role in asthma. However, scarce information exists about the pulmonary expression of 5-HT receptors and its modification after allergic sensitization. In the present work, we explored the expression of 5-HT1A, 5-HT2A, 5-HT3, 5-HT4, 5-ht5a, 5-HT6, and 5-HT7 receptors in lungs from control and sensitized guinea pigs through qPCR and Western blot. In control animals, mRNA from all receptors was detectable in lung homogenates, especially from 5-HT2A and 5-HT4 receptors. Sensitized animals had decreased mRNA expression of 5-HT2A and 5-HT4 receptors and increased that of 5-HT7 receptor. In contrast, they had increased protein expression of 5-HT2A receptor in bronchial epithelium and of 5-HT4 receptor in lung parenchyma. The degree of airway response to the allergic challenge was inversely correlated with mRNA expression of the 5-HT1A receptor. In summary, our results showed that major 5-HT receptor subtypes are constitutively expressed in the guinea pig lung, and that allergic sensitization modifies the expression of 5-HT2A, 5-HT4, and 5-HT7 receptors. PMID:26657047

  12. Differential expression of genes for aromatase and estrogen receptor during the gonadal development in chicken embryos.

    PubMed

    Nakabayashi, O; Kikuchi, H; Kikuchi, T; Mizuno, S

    1998-04-01

    In birds, differentiation of embryonic gonads is not as strictly determined by the genetic sex as it is in mammals, and can be influenced by early manipulation with a sex steroid hormone. Thus administration of an aromatase inhibitor induces testis development in the genetic female, and administration of estrogen induces a left ovotestis in the genetic male embryo. Another feature of avian gonadogenesis is that only the left ovary develops in most species. Molecular mechanisms underlying these features at the level of gene expression have not been elucidated. In this paper, we present evidence that a gene for aromatase cytochrome P-450, an enzyme required for the last step in the synthesis of estradiol-17beta, is expressed in medullae of the left and right gonads of a female chicken embryo, but not in those of a male chicken embryo, and that an estrogen receptor gene is expressed only in epithelium (and cortex later, in the female) of the left, not the right, gonad of both sexes, but the expression in the male left gonad is temporary and restricted to an early stage of development. Differential expression of these two genes serves well to explain the above features of gonadal development in birds. Furthermore, in ovo administration of estradiol-17beta from the 5th to the 14th day of incubation does not cause expression of the estrogen receptor gene in the right gonad of chicken embryos of either sex, suggesting that the absence of expression of the estrogen receptor gene in the right gonad is not the result of down-regulation, but may be regarded as an important cause of the unilateral ovarian development. PMID:9584834

  13. Dynamic Regulation of Platelet-Derived Growth Factor Receptor α Expression in Alveolar Fibroblasts during Realveolarization

    PubMed Central

    Chen, Leiling; Acciani, Thomas; Le Cras, Tim; Lutzko, Carolyn

    2012-01-01

    Although the importance of platelet-derived growth factor receptor (PDGFR)-α signaling during normal alveogenesis is known, it is unclear whether this signaling pathway can regulate realveolarization in the adult lung. During alveolar development, PDGFR-α–expressing cells induce α smooth muscle actin (α-SMA) and differentiate to interstitial myofibroblasts. Fibroblast growth factor (FGF) signaling regulates myofibroblast differentiation during alveolarization, whereas peroxisome proliferator-activated receptor (PPAR)-γ activation antagonizes myofibroblast differentiation in lung fibrosis. Using left lung pneumonectomy, the roles of FGF and PPAR-γ signaling in differentiation of myofibroblasts from PDGFR-α–positive precursors during compensatory lung growth were assessed. FGF receptor (FGFR) signaling was inhibited by conditionally activating a soluble dominant-negative FGFR2 transgene. PPAR-γ signaling was activated by administration of rosiglitazone. Changes in α-SMA and PDGFR-α protein expression were assessed in PDGFR-α–green fluorescent protein (GFP) reporter mice using immunohistochemistry, flow cytometry, and real-time PCR. Immunohistochemistry and flow cytometry demonstrated that the cell ratio and expression levels of PDGFR-α–GFP changed dynamically during alveolar regeneration and that α-SMA expression was induced in a subset of PDGFR-α–GFP cells. Expression of a dominant-negative FGFR2 and administration of rosiglitazone inhibited induction of α-SMA in PDGFR-α–positive fibroblasts and formation of new septae. Changes in gene expression of epithelial and mesenchymal signaling molecules were assessed after left lobe pneumonectomy, and results demonstrated that inhibition of FGFR2 signaling and increase in PPAR-γ signaling altered the expression of Shh, FGF, Wnt, and Bmp4, genes that are also important for epithelial–mesenchymal crosstalk during early lung development. Our data demonstrate for the first time that a comparable

  14. Estrogen Receptor Beta Expression in the Mouse Forebrain: Age and Sex Differences

    PubMed Central

    Zuloaga, Damian G.; Zuloaga, Kristen L.; Hinds, Laura R.; Carbone, David L.; Handa, Robert J.

    2016-01-01

    Estrogen receptors regulate multiple brain functions including stress, sexual, and memory associated behaviors as well as control of neuroendocrine and autonomic function. During development, estrogen signaling is involved in programming adult sex differences in physiology and behavior. Expression of estrogen receptor alpha changes across development in a region specific fashion. By contrast, estrogen receptor beta (ERβ) is expressed in many brain regions, yet few studies have explored sex and developmental differences in its expression largely due to the absence of selective reagents for anatomical localization of the protein. In this study, we utilized bacterial artificial chromosome transgenic mice expressing ERβ identified by enhanced green fluorescent protein (EGFP) to compare expression levels and distribution of ERβ in the male and female mouse forebrain on the day of birth (P0), postnatal day 4 (P4) and P21. Using qualitative analysis, we mapped the distribution of ERβ–EGFP and found developmental alterations in ERβ expression within the cortex, hippocampus, and hypothalamic regions including the arcuate, ventromedial, and paraventricular nuclei. We also report a sex difference in ERβ in the bed nucleus of the stria terminalis with males showing greater expression at P4 and P21. Another sex difference was found in the anteroventral periventricular nucleus of P21, but not P0 or P4 mice, where ERβ-EGFP-ir cells were densely clustered near the 3rd ventricle in females but not males. These developmental changes and sex differences in ERβ indicate a mechanism through which estrogens may differentially affect brain functions or program adult physiology at select times during development. PMID:23818057

  15. Effects of a Levonorgestrel-Releasing Intrauterine System on the Expression of Steroid Receptor Coregulators in Adenomyosis.

    PubMed

    Yun, Bo Hyon; Jeon, Young Eun; Seo, Seok Kyo; Park, Joo Hyun; Yoon, Sun Och; Cho, SiHyun; Choi, Young Sik; Lee, Byung Seok

    2015-12-01

    Although the pathophysiology of adenomyosis has not been clarified, it is thought to be related to ectopic endometrium, which depends on hormonal regulation. The levonorgestrel-releasing intrauterine system (LNG-IUS) is effective for the medical treatment of adenomyosis. However, the underlying molecular mechanisms by which LNG-IUS ameliorates adenomyosis pathology remain unclear. This study was designed to compare the expression levels of steroid receptor coregulators in human endometrium of control and participants with adenomyosis and to determine whether LNG-IUS modulated their expression. Immunohistochemistry with H-scores was performed. Steroid receptor coactivators were shown to have significantly decreased expressions at the secretory phase in the LNG-IUS group when compared to the other groups. Expression of transcriptional intermediary factor 2 was lower in the LNG-IUS group than in both the control group (P = .015) and the untreated adenomyosis group (P = .019) during the secretory phase. Amplified in breast cancer 1 expression was higher in the stromal cells of the untreated adenomyosis group than in those of the controls (P = .017) during the secretory phase; however, levels were lower in the LNG-IUS group (P = .005). Nuclear receptor corepressor expression increased during the proliferative phase and decreased during the secretory phase in untreated adenomyosis; this pattern was reversed in the control and LNG-IUS groups. Thus, an altered expression of steroid receptor coregulators may play a role in adenomyosis development and treatment. PMID:26040939

  16. Homologous upregulation of sst2 somatostatin receptor expression in the rat arcuate nucleus in vivo.

    PubMed

    Tannenbaum, G S; Turner, J; Guo, F; Videau, C; Epelbaum, J; Beaudet, A

    2001-07-01

    In vitro studies using various cell systems have provided conflicting results regarding homologous regulation of somatostatin (SRIH) receptors, and information on whether SRIH regulates the expression of its own receptors in vivo is lacking. In the present study we examined, by in situ hybridization, the effects of pretreatment with the sst2-preferring SRIH analog, octreotide, in vivo, on mRNA levels of two SRIH receptor subtypes, sst1 and sst2, in rat brain and pituitary. (125)I-[DTrp(8)]-SRIH binding was also measured in these regions. Three hours after the iv injection of 50 microg octreotide to conscious adult male rats, there was a 46% increase (p < 0.01) in the labeling density of sst2 mRNA-expressing cells in the hypothalamic arcuate nucleus compared to normal saline-pretreated controls, but not in any of the other brain regions examined. Computer-assisted image analysis revealed that 3 h exposure to octreotide significantly (p < 0.01) augmented both the number and labeling density of sst2 mRNA-expressing cells in the arcuate nucleus, compared to those in saline-treated controls. By contrast, within the anterior pituitary gland, in vivo exposure to octreotide did not affect the expression of sst2 mRNA. No changes in sst1 mRNA-expressing cells were observed after octreotide treatment in any of the regions measured, indicating that the observed effects were homologous, i.e. specific of the receptor subtype stimulated. Octreotide pretreatment was also without effect on the density of (125)I-[DTrp(8)]-SRIH binding in either the arcuate nucleus or pituitary. These results demonstrate, for the first time, that SRIH preexposure in vivo upregulates the expression of a subtype of its own receptors, sst2, within the central nervous system. They further suggest that pretreatment with SRIH in vivo does not cause sst2 receptor desensitization in arcuate nucleus and pituitary. Such homologous regulatory mechanisms may play an important role in the neuroendocrine control

  17. The Aryl Hydrocarbon Receptor Complex and the Control of Gene Expression

    PubMed Central

    Beischlag, Timothy V.; Morales, J. Luis; Hollingshead, Brett D.; Perdew, Gary H.

    2008-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that controls the expression of a diverse set of genes. The toxicity of the potent AhR ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin is almost exclusively mediated through this receptor. However, the key alterations in gene expression that mediate toxicity are poorly understood. It has been established through characterization of AhR-null mice that the AhR has a required physiological function, yet how endogenous mediators regulate this orphan receptor remains to be established. A picture as to how the AhR/ARNT heterodimer actually mediates gene transcription is starting to emerge. The AhR/ARNT complex can alter transcription both by binding to its cognate response element and through tethering to other transcription factors. In addition, many of the coregulatory proteins necessary for AhR-mediated transcription have been identified. Cross talk between the estrogen receptor and the AhR at the promoter of target genes appears to be an important mode of regulation. Inflammatory signaling pathways and the AhR also appear to be another important site of cross talk at the level of transcription. A major focus of this review is to highlight experimental efforts to characterize nonclassical mechanisms of AhR-mediated modulation of gene transcription. PMID:18540824

  18. Pentadecapeptide BPC 157 enhances the growth hormone receptor expression in tendon fibroblasts.

    PubMed

    Chang, Chung-Hsun; Tsai, Wen-Chung; Hsu, Ya-Hui; Pang, Jong-Hwei Su

    2014-01-01

    BPC 157, a pentadecapeptide derived from human gastric juice, has been demonstrated to promote the healing of different tissues, including skin, muscle, bone, ligament and tendon in many animal studies. However, the underlying mechanism has not been fully clarified. The present study aimed to explore the effect of BPC 157 on tendon fibroblasts isolated from Achilles tendon of male Sprague-Dawley rat. From the result of cDNA microarray analysis, growth hormone receptor was revealed as one of the most abundantly up-regulated genes in tendon fibroblasts by BPC 157. BPC 157 dose- and time-dependently increased the expression of growth hormone receptor in tendon fibroblasts at both the mRNA and protein levels as measured by RT/real-time PCR and Western blot, respectively. The addition of growth hormone to BPC 157-treated tendon fibroblasts dose- and time-dependently increased the cell proliferation as determined by MTT assay and PCNA expression by RT/real-time PCR. Janus kinase 2, the downstream signal pathway of growth hormone receptor, was activated time-dependently by stimulating the BPC 157-treated tendon fibroblasts with growth hormone. In conclusion, the BPC 157-induced increase of growth hormone receptor in tendon fibroblasts may potentiate the proliferation-promoting effect of growth hormone and contribute to the healing of tendon. PMID:25415472

  19. Regulation of the surface expression of the platelet-activating factor receptor in IC-21 peritoneal macrophages. Effects of lipopolysaccharide.

    PubMed

    Liu, H; Chao, W; Olson, M S

    1992-10-15

    The effect of bacterial lipopolysaccharide (LPS) on the expression of the receptor for platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; AGEPC) was examined in cultured IC-21 peritoneal macrophages. AGEPC binding to its receptors reached saturation within 20 min at 25 degrees C and was reversible. Scatchard analysis revealed a single class of AGEPC receptors with a Bmax of approximately 170 fmol/mg cellular protein and a Kd of 0.25 nM. Preincubation of IC-21 cells with LPS (0.01-1,000 ng/ml) induced an increase in the surface expression of AGEPC receptors in a time- and concentration-dependent fashion. The maximal effect of LPS on the AGEPC receptor was observed between 5 and 8 h, with a typical increase between 150 and 200%. Scatchard analysis indicated that LPS treatment of IC-21 cells increased the number of AGEPC receptors on the cell surface without any apparent change in the affinity of the receptor for the ligand. The effect of LPS on the surface expression of the AGEPC receptor was nearly abolished by cycloheximide (0.1 mM) and by actinomycin D (3 microM), suggesting the involvement of enhanced receptor protein synthesis and mRNA production in this event. Moreover, LPS treatment increased the capability of the IC-21 cell to respond to AGEPC addition by elevating intracellular free Ca2+ without causing an increase in the basal level of intracellular Ca2+. The present study demonstrates that IC-21 peritoneal macrophages possess high affinity AGEPC receptors and provides the evidence that the number of functional AGEPC receptors on a cell can be increased significantly upon exposure to LPS. PMID:1328211

  20. Disruption in the expression and immunolocalisation of steroid receptors and steroidogenic enzymes in letrozole-induced polycystic ovaries in rat.

    PubMed

    Zurvarra, Francisco M; Salvetti, Natalia R; Mason, J Ian; Velazquez, Melisa M L; Alfaro, Natalia S; Ortega, Hugo H

    2009-01-01

    The objective of the present study was to characterise the expression and tissue distribution of steroid receptors (oestrogen receptor-alpha and -beta (ERalpha, ERbeta), androgen receptor (AR) and progesterone receptor (PR)) and steroidogenic enzymes (P450 aromatase (P450arom), 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and steroidogenic acute regulatory protein (StAR)) in letrozole-induced polycystic ovaries of rats. Changes in serum hormone levels, protein expression in whole ovaries by western blot analysis and protein localisation by immunohistochemistry were determined in female rats treated with the aromatase inhibitor letrozole and compared with controls in proestrous and diestrous rats. Increases in the serum LH, FSH and testosterone concentrations were observed in letrozole-treated rats whereas serum oestradiol and progesterone levels were reduced. Protein expression as analysed by western immunoblot was consistent with the immunohistochemical data. Letrozole treatment induced an increase in the expression of AR, StAR and 3beta-HSD and a decrease in ERbeta. ERalpha, PR and P450arom showed partial changes in relation to some cycle stages. These results indicate that cystogenesis in this experimental model is characterised by changes in steroid receptors and steroidogenic enzyme expression that may be essential to proper ovarian functioning and are in agreement with similar changes observed in women with PCOS. PMID:19698287

  1. Tissue plasminogen activator inhibits NMDA-receptor-mediated increases in calcium levels in cultured hippocampal neurons

    PubMed Central

    Robinson, Samuel D.; Lee, Tet Woo; Christie, David L.; Birch, Nigel P.

    2015-01-01

    NMDA receptors (NMDARs) play a critical role in neurotransmission, acting as essential mediators of many forms of synaptic plasticity, and also modulating aspects of development, synaptic transmission and cell death. NMDAR-induced responses are dependent on a range of factors including subunit composition and receptor location. Tissue-type plasminogen activator (tPA) is a serine protease that has been reported to interact with NMDARs and modulate NMDAR activity. In this study we report that tPA inhibits NMDAR-mediated changes in intracellular calcium levels in cultures of primary hippocampal neurons stimulated by low (5 μM) but not high (50 μM) concentrations of NMDA. tPA also inhibited changes in calcium levels stimulated by presynaptic release of glutamate following treatment with bicucculine/4-aminopyridine (4-AP). Inhibition was dependent on the proteolytic activity of tPA but was unaffected by α2-antiplasmin, an inhibitor of the tPA substrate plasmin, and receptor-associated protein (RAP), a pan-ligand blocker of the low-density lipoprotein receptor, two proteins previously reported to modulate NMDAR activity. These findings suggest that tPA can modulate changes in intracellular calcium levels in a subset of NMDARs expressed in cultured embryonic hippocampal neurons through a mechanism that involves the proteolytic activity of tPA and synaptic NMDARs. PMID:26500501

  2. High Cell Surface Death Receptor Expression Determines Type I Versus Type II Signaling*

    PubMed Central

    Meng, Xue Wei; Peterson, Kevin L.; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D.; Gores, Gregory J.; Kaufmann, Scott H.

    2011-01-01

    Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression. PMID:21865165

  3. Differential relationships between D1 and D2 dopamine receptor expression in the medial preoptic nucleus and sexually-motivated song in male European starlings (Sturnus vulgaris).

    PubMed

    DeVries, M S; Cordes, M A; Stevenson, S A; Riters, L V

    2015-08-20

    Converging data in songbirds support a central role for the medial preoptic nucleus (POM) in motivational aspects of vocal production. Recent data suggest that dopamine in the POM plays a complex modulatory role in the production of sexually-motivated song and that an optimal level of dopamine D1 receptor stimulation is required to facilitate singing behavior. To further explore this possibility, we used quantitative real-time PCR to examine relationships between mRNA expression of D1 as well as D2 receptors in the POM (and also the lateral septum and Area X) and sexually-motivated singing behavior in male European starlings. Results showed that both males with the highest and lowest D1 expression in the POM sang significantly less than males with intermediate levels of expression. Furthermore, singing behavior rose linearly in association with increasing levels of D1 expression in POM but dropped abruptly, such that individuals with D1 expression values higher than the mean sang very little. Analysis of birds with low and intermediate levels of D1 expression in POM revealed strong positive correlations between D1 expression and song but negative relationships between D2 receptor expression and song. These findings support prior work suggesting an optimal level of POM D1 receptor stimulation best facilitates sexually-motivated singing behavior. Results also suggest that D2 receptors may work in opposition to D1 receptors in POM to modify vocal production. PMID:26079111

  4. Behavior of a cloned murine interferon alpha/beta receptor expressed in homospecific or heterospecific background.

    PubMed Central

    Uzé, G; Lutfalla, G; Bandu, M T; Proudhon, D; Mogensen, K E

    1992-01-01

    A murine interferon (IFN) alpha/beta receptor was cloned from the IFN-sensitive L1210 cell line on the basis of its homology with the human receptor. A combination of methods that includes the screening of random-primed and oligo(dT)-primed cDNA libraries and polymerase chain reactions with a single-side specificity was used. At the amino acid level, the murine IFN-alpha/beta shows 46% identity with its human counterpart. Both human WISH cells presenting a low sensitivity to mouse IFN and a murine L1210 mutant subline that does not express the receptor have been stably transfected with the murine IFN-alpha/beta receptor. Whereas transfected human cells became sensitive to a limited number of mouse IFN-alpha/beta subtypes, the transfected murine L1210 mutant was found to be fully complemented and became sensitive to all mouse IFN-alpha/beta subtypes tested, including those that were not active on transfected human cells. These results strongly suggest that the receptor described here is implicated in the mediation of the activities of all murine IFN-alpha/beta subtypes. Images PMID:1533935

  5. Relationship between circadian oscillations of Rev-erb{alpha} expression and intracellular levels of its ligand, heme

    SciTech Connect

    Rogers, Pamela M.; Ying Ling; Burris, Thomas P.

    2008-04-18

    The nuclear hormone receptors, REV-ERB{alpha} [NR1D1] and REV-ERB{beta} [NR1D1], were recently demonstrated to be receptors for the porphyrin, heme. Heme regulates the ability of these receptors to repress transcription of their target genes via modulation of the affinity of the receptor's ligand binding domain for the corepressor, NCoR. The REV-ERBs function as critical components of the mammalian clock and their expression oscillates in a circadian manner. Here, we show that in NIH3T3 cells intracellular heme levels also oscillate in a circadian fashion. These data are the first to show the temporal relationship of intracellular heme levels to the expression of its receptor, Rev-erb{alpha}, and suggest that the rapid oscillations in heme levels may an important component regulating REV-ERB transcriptional activity.

  6. Expression of histamine H4 receptor in human epidermal tissues and attenuation of experimental pruritus using H4 receptor antagonist.

    PubMed

    Yamaura, Katsunori; Oda, Manabu; Suwa, Eriko; Suzuki, Masahiko; Sato, Hiromi; Ueno, Koichi

    2009-10-01

    Many medicines exist which can cause pruritus (itching) as "serious adverse events." Many severe pruritic conditions respond poorly to histamine H1 receptor antagonists; there is no generally accepted antipruritic treatment. Recently described histamine H4 receptors are expressed in haematopoietic cells and have been linked to the pathology of allergy and asthma. We previously reported their expression in human dermal fibroblasts; in this study we have investigated H4 receptor expression in human epidermal tissue and found it to be greater in keratinocytes in the epidermal upper layer than in the lower layer. We have also investigated the effect of histamine H4 receptor antagonists on histamine H1 receptor antagonist-resistant pruritus using a mouse model. Scratching behavior was induced by histamine (300 nmol) or substance P (100 nmol) injected intradermally into the rostral part of the back of each mouse. Fexofenadine, a histamine H1 receptor antagonist, reduced scratching induced by histamine but not by substance P, whereas JNJ7777120, a histamine H4 receptor antagonist, significantly reduced both histamine- and substance P-induced scratching. These results suggest that H4 receptor antagonists may be useful for treatment of H1 receptor antagonist-resistant pruritus. PMID:19652466

  7. A study protocol for quantitative targeted absolute proteomics (QTAP) by LC-MS/MS: application for inter-strain differences in protein expression levels of transporters, receptors, claudin-5, and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice

    PubMed Central

    2013-01-01

    Proteomics has opened a new horizon in biological sciences. Global proteomic analysis is a promising technology for the discovery of thousands of proteins, post-translational modifications, polymorphisms, and molecular interactions in a variety of biological systems. The activities and roles of the identified proteins must also be elucidated, but this is complicated by the inability of conventional proteomic methods to yield quantitative information for protein expression. Thus, a variety of biological systems remain “black boxes”. Quantitative targeted absolute proteomics (QTAP) enables the determination of absolute expression levels (mol) of any target protein, including low-abundance functional proteins, such as transporters and receptors. Therefore, QTAP will be useful for understanding the activities and roles of individual proteins and their differences, including normal/disease, human/animal, or in vitro/in vivo. Here, we describe the study protocols and precautions for QTAP experiments including in silico target peptide selection, determination of peptide concentration by amino acid analysis, setup of selected/multiple reaction monitoring (SRM/MRM) analysis in liquid chromatography–tandem mass spectrometry, preparation of protein samples (brain capillaries and plasma membrane fractions) followed by the preparation of peptide samples, simultaneous absolute quantification of target proteins by SRM/MRM analysis, data analysis, and troubleshooting. An application of QTAP in biological sciences was introduced that utilizes data from inter-strain differences in the protein expression levels of transporters, receptors, tight junction proteins and marker proteins at the blood–brain barrier in ddY, FVB, and C57BL/6J mice. Among 18 molecules, 13 (abcb1a/mdr1a/P-gp, abcc4/mrp4, abcg2/bcrp, slc2a1/glut1, slc7a5/lat1, slc16a1/mct1, slc22a8/oat3, insr, lrp1, tfr1, claudin-5, Na+/K+-ATPase, and γ-gtp) were detected in the isolated brain capillaries, and their

  8. Involvement of 5-HT₃ and 5-HT₄ receptors in the regulation of circadian clock gene expression in mouse small intestine.

    PubMed

    Aoki, Natsumi; Watanabe, Hiroyuki; Okada, Kazuya; Aoki, Kazuyuki; Imanishi, Takuma; Yoshida, Daisuke; Ishikawa, Ryosuke; Shibata, Shigenobu

    2014-01-01

    Several lines of evidence suggest that 5-HT receptors play a critical role in the expression of clock genes in the suprachiasmatic nucleus, the main circadian oscillator in hamsters. The contributions of 5-HT-receptor subtypes in the intestine, where they are expressed at high concentrations, are however not yet clarified. The 5-HT synthesis inhibitor, p-chlorophenylalanine, attenuated the daily rhythm of Per1 and Per2 gene expression in the intestine. Injection of 5-HT and agonists of the 5-HT3 and 5-HT4 receptors increased Per1/Per2 expression and decreased Bmal1 expression in a dose-dependent manner. Although treatment with antagonists of 5-HT3 and 5-HT4 alone did not affect clock gene expression, co-injection of these antagonists with 5-HT blocked the 5-HT-induced changes in clock gene expression. Increased tissue levels of 5-HT due to treatment with the antidepressants clomipramine and fluvoxamine did not affect clock gene expression. The present results suggest that the 5-HT system in the small intestine may play a critical role in regulating circadian rhythms through 5-HT3/5-HT4-receptor activation. PMID:24492464

  9. Molecular cloning, characterization, and expression analysis of an ecdysone receptor homolog in Teleogryllus emma (Orthoptera: Gryllidae).

    PubMed

    He, Hui; Xi, Gengsi; Lu, Xiao

    2015-01-01

    Ecdysteroids are steroid hormones that play important roles in the regulation of Arthropoda animal growth development, larvae ecdysis, and reproduction. The effect of ecdysteroids is mediated by ecdysteroid receptor (EcR). The ecdysone receptor (EcR) belongs to the superfamily of nuclear receptors (NRs) that are ligand-dependent transcription factors. Ecdysone receptor is present only in invertebrates and plays a critical role in regulating the expression of a series of genes during development and reproduction. Here, we isolated and characterized cDNA of the cricket Teleopgryllus emma (Ohmachi & Matsuura) (Orthoptera: Gryllidae) and studied mRNA expression pattern using real time-polymerase chain reaction. The full-length cDNA of T. emma EcR, termed TeEcR, is 2,558 bp and contains a 5'-untranslated region of 555 bp and a 3'-untranslated region of 407 bp. The open reading frame of TeEcR encodes deduced 531-amino acid peptides with a predicted molecular mass of 60.7 kDa. The amino acid sequence of T. emma EcR was similar to that of known EcR especially in the ligand-binding domain of insect EcR. Real-time quantitative reverse transcription-polymerase chain reaction was performed to compare TeEcR mRNA expression level at the whole body and gonad during T. emma development. The data revealed that TeEcR mRNA is differentially expressed during T. emma development, with the highest expression level in late-instar larvae of the body and lowest in third instar. The levels of TeEcR transcripts also vary among gonads development, and levels in ovaries were higher than in testes at every developmental stage. These results suggest that TeEcR may have potential significance to regulate the morphological structure and gonad development of T. emma, due to its expression in different developmental periods. PMID:25797799