Hormonal and environmental regulation of epithelial calcium channel in gill of rainbow trout (Oncorhynchus mykiss)

2006 ◽  
Vol 291 (5) ◽  
pp. R1490-R1498 ◽  
Author(s):  
Arash Shahsavarani ◽  
Steve F. Perry
Keyword(s):  
Protein Expression ◽  
Fold Increase ◽  
Mrna Levels ◽  
Uptake Capacity ◽  
Pavement Cells ◽  
Protein Levels ◽  
Rainbow Trout Oncorhynchus Mykiss ◽  
Uptake Rates ◽  
Mrna And Protein Expression ◽  
Trout Gill

We indirectly tested the idea that the epithelial Ca2+ channel (ECaC) of the trout gill is regulated in an appropriate manner to adjust rates of Ca2+ uptake. This was accomplished by assessing the levels of gill ECaC mRNA and protein in fish exposed to treatments known to increase or decrease Ca2+ uptake capacity. Exposure of trout to soft water ([Ca2+] = 20–30 nmol/l) for 5 days (a treatment known to increase Ca2+ uptake capacity) caused a significant increase in ECaC mRNA levels and an increase in ECaC protein expression. The inducement of hypercalcemia by infusing fish with CaCl2 (a treatment known to reduce Ca2+ uptake) was associated with a significant decrease in ECaC mRNA levels, yet protein levels were unaltered. ECaC mRNA and protein expression were increased in fish treated with the hypercalcemic hormone cortisol. Finally, exposure of trout to 48 h of hypercapnia (∼7.5 mmHg, a treatment known to increase Ca2+ uptake capacity) elicited an ∼100-fold increase in the levels of ECaC mRNA and a significant increase in protein expression. Immunocytochemical analysis of the gills from hypercapnic fish suggested a marked increase in the apical expression of ECaC on pavement cells and a subpopulation of mitochondria-rich cells. The results of this study provide evidence that Ca2+ uptake rates are, in part, regulated by the numbers of apical membrane Ca2+ channels that, in turn, modulate the inward flux of Ca2+ into gill epithelial cells.

scholarly journals Yiqi Huoxue Recipe Improves Liver Regeneration in Rats after Partial Hepatectomy via JNK Pathway

2020 ◽  
Vol 2020 ◽  
pp. 1-9
Author(s):  
Wen-cong Li ◽  
Su-xian Zhao ◽  
Wei-guang Ren ◽  
Hui-juan Du ◽  
Yu-guo Zhang ◽  
...  
Keyword(s):  
Protein Expression ◽  
Liver Regeneration ◽  
Partial Hepatectomy ◽  
Underlying Mechanism ◽  
Mrna Levels ◽  
Protective Effects ◽  
Jnk Pathway ◽  
Expression Levels ◽  
Protein Levels ◽  
Mrna And Protein Expression

The liver is the only visceral organ that exhibits a remarkable capability of regenerating in response to partial hepatectomy (PH) or chemical injury. Improving liver regeneration (LR) ability is the basis for the favourable treatment outcome of patients after PH, which can serve as a potential indicator for postoperative survival. The present study aimed to investigate the protective effects of Yiqi Huoxue recipe (YQHX) on LR after PH in rats and further elucidate its underlying mechanism. A two-thirds PH rat model was used in this study. Wistar rats were randomly divided into four groups: sham-operated, PH, YQHX + PH, and Fuzheng Huayu decoction (FZHY) + PH groups. All rats were sacrificed under anesthesia at 24 and 72 h after surgery. The rates of LR were calculated, and the expression levels of cyclin D1 and c-jun were determined by immunohistochemical staining. The protein levels of p-JNK1/2, JNK1/2, p-c-jun, c-jun, Bax, and Bcl-2 were detected by Western blotting, while the mRNA levels of JNK1, JNK2, c-jun, Bax, and Bcl-2 were examined by real-time polymerase chain reaction (RT-PCR). At the corresponding time points, YQHX and FZHY administration dramatically induced the protein levels of p-JNK1/2 compared to the PH group p<0.05, while FZHY + PH group showed prominently increase in p-JNK1/2 protein levels compared to the YQHX + PH group p<0.05. A similar trend was observed for the expression levels of p-c-jun. Compared to the PH group, YQHX and FZHY markedly reduced the mRNA and protein expression levels of Bax at 24 h after PH, while those in the FZHY + PH group decreased more obviously p<0.05. Besides, in comparison with the PH group, YQHX and FZHY administration predominantly upregulated the mRNA and protein expression levels of Bcl-2 at 24 and 72 h after PH p<0.05. In conclusion, YQHX improves LR in rats after PH by inhibiting hepatocyte apoptosis via the JNK signaling pathway.

scholarly journals Polymorphism in the progesterone receptor promoter gene in endometrial cancer alters its expression

2020 ◽  
Author(s):  
Zhengwu Xiao ◽  
Huahua Xiang ◽  
Jing Zhou ◽  
Chen Zhou ◽  
Zifen Guo
Keyword(s):  
Endometrial Cancer ◽  
Progesterone Receptor ◽  
Protein Expression ◽  
Cancer Patients ◽  
Control Group ◽  
Mrna Levels ◽  
Normal Endometrium ◽  
Protein Levels ◽  
Healthy Control ◽  
Mrna And Protein Expression

Abstract The purpose of this study was to investigate the effect of progesterone receptor (PGR) promoter +331G/A polymorphism on the mRNA and protein expression of its two isoforms, PRA and PRB, in healthy control women and women with endometrial cancer. To evaluate the relative occurrence of +331G/A polymorphism, the PGR gene promoter in the whole blood of 66 healthy volunteers and 62 endometrial cancer patients was genotyped. The results demonstrate that the frequency of GG and the overall frequency of the G allele were >90% in both populations. The GA+AA genotypes were more common in the healthy control group than in the endometrial cancer group, though the differences were not statistically significant. RT-PCR and Western blot analysis results showed that the mRNA and protein levels of both PRB and PRA were significantly lower in endometrium from cancer patients than in normal endometrium tissue. Furthermore, among individuals with endometrial cancer, those with the +331 G/A polymorphism expressed higher mRNA levels of the PRA isoform and higher protein levels of the PRB isoform. Therefore, our findings suggest that patients with endometrial cancer express less PGR and that the mRNA and protein expression of PRA and PRB may be altered due to 331G/A PGR gene polymorphism.

Synthesis of leukotrienes in porcine uteri with endometritis induced by infection with Escherichia coli

2014 ◽  
Vol 26 (7) ◽  
pp. 1007 ◽  
Author(s):  
Barbara Jana ◽  
Joanna Czarzasta ◽  
Jerzy Jaroszewski
Keyword(s):  
Escherichia Coli ◽  
Protein Expression ◽  
Significant Role ◽  
Present Experiment ◽  
Inflammatory Process ◽  
Lipid Mediators ◽  
Mrna Levels ◽  
Protein Levels ◽  
Uterine Inflammation ◽  
Mrna And Protein Expression

Leukotrienes (LTs) are lipid mediators that play a significant role in the inflammatory process. Their production in inflamed uteri is not fully understood. The present experiment aimed to determine LTB4 and LTC4 amounts, 5-lipooxygenase (5-LO), LTA4 hydrolase (LTAH) and LTC4 synthase (LTCS) mRNA levels and protein expression in inflamed porcine uteri. On Day 3 of the oestrous cycle (Day 0 of the study), either Escherichia coli suspension or saline were infused into uterine horns. Collection of uterine tissues and washings took place eight or sixteen days later. In gilts suffering from endometritis increased LTB4 and LTC4 levels in the endometrium and washings and 5-LO mRNA levels in the myometrium on Days 8 and 16, 5-LO protein levels in the endometrium and myometrium on Day 8, LTAH mRNA and protein levels in the endometrium and myometrium on Days 8 and 16, respectively. Although LTCS mRNA and protein expression in the myometrium and LTCS protein expression in the endometrium were enhanced on Day 16 after Escherichia coli inoculation, LTCS mRNA levels decreased on Day 8 in both tissues. Our study shows the upregulation of LT production in inflamed porcine uteri, which suggests the importance of these factors to the process of uterine inflammation.

scholarly journals Bovine gonadotrophs express anti-Müllerian hormone (AMH): comparison of AMH mRNA and protein expression levels between old Holsteins and young and old Japanese Black females

2019 ◽  
Vol 31 (4) ◽  
pp. 810 ◽  
Author(s):  
Onalenna Kereilwe ◽  
Hiroya Kadokawa
Keyword(s):  
Protein Expression ◽  
Mrna Levels ◽  
Expression Levels ◽  
Black Females ◽  
Protein Levels ◽  
Gonadotrophin Secretion ◽  
Old Japanese ◽  
Mrna And Protein Expression ◽  
Gonadotrophin Releasing Hormone ◽  
Polymerase Chain

Anti-Müllerian hormone (AMH) is secreted from ovaries and stimulates gonadotrophin secretion from bovine gonadotroph cells. Other important hormones for endocrinological gonadotroph regulation (e.g. gonadotrophin-releasing hormone, inhibin and activin) have paracrine and autocrine roles. Therefore, in this study, AMH expression in bovine gonadotroph cells and the relationships between AMH expression in the bovine anterior pituitary (AP) and oestrous stage, age and breed were evaluated. AMH mRNA expression was detected in APs of postpubertal heifers (26 months old) by reverse transcription-polymerase chain reaction. Based on western blotting using an antibody to mature C-terminal AMH, AMH protein expression was detected in APs. Immunofluorescence microscopy utilising the same antibody indicated that AMH is expressed in gonadotrophs. The expression of AMH mRNA and protein in APs did not differ between oestrous phases (P&gt;0.1). We compared expression levels between old Holsteins (79.2±10.3 months old) and young (25.9±0.6 months old) and old Japanese Black females (89.7±20.3 months old). The APs of old Holsteins exhibited lower AMH mRNA levels (P&lt;0.05) but higher AMH protein levels than those of young Japanese Black females (P&lt;0.05). In conclusion, bovine gonadotrophs express AMH and this AMH expression may be breed-dependent.

Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium

1997 ◽  
Vol 273 (6) ◽  
pp. C1937-C1946 ◽  
Author(s):  
James F. Collins ◽  
Hua Xu ◽  
Pawel R. Kiela ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Polyclonal Antibodies ◽  
Membrane Vesicle ◽  
Age Groups ◽  
Fold Increase ◽  
Jejunal Mucosa ◽  
Mrna Levels ◽  
Adult Rats ◽  
Intestinal Brush Border Membrane ◽  
Protein Levels

Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 μM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant ( K i) = 5 μM in PS120 fibroblasts] and NHE3 ( K i = 650 μM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 μM HOE-694 was 38–45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.

scholarly journals Regulation of AMH by oocyte-specific growth factors in human primary cumulus cells

Reproduction ◽  
2017 ◽  
Vol 154 (6) ◽  
pp. 745-753 ◽  
Author(s):  
Scott Convissar ◽  
Marah Armouti ◽  
Michelle A Fierro ◽  
Nicola J Winston ◽  
Humberto Scoccia ◽  
...  
Keyword(s):  
Fold Increase ◽  
Cumulus Cells ◽  
Maximal Effect ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Protein Levels ◽  
Secreted Factors ◽  
Morphogenetic Protein ◽  
Growth Differentiation Factor 9 ◽  
Signaling Inhibitors

The regulation of AMH production by follicular cells is poorly understood. The purpose of this study was to determine the role of the oocyte-secreted factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on AMH production in primary human cumulus cells. Cumulus cells from IVF patients were cultured with a combination of GDF9, BMP15, recombinant FSH and specific signaling inhibitors. Stimulation with GDF9 or BMP15 separately had no significant effect onAMHmRNA levels. In contrast, simultaneous stimulation with GDF9 and BMP15 (G + B) resulted in a significant increase inAMHmRNA expression. Increasing concentration of G + B (0.6, 2.5, 5 and 10 ng/mL) stimulated AMH in a dose-dependent manner, showing a maximal effect at 5 ng/mL. Western blot analyses revealed an average 16-fold increase in AMH protein levels in cells treated with G + B when compared to controls. FSH co-treatment decreased the stimulation of AMH expression by G + B. The stimulatory effect of G + B on the expression of AMH was significantly decreased by inhibitors of the SMAD2/3 signaling pathway. These findings show for the first time that AMH production is regulated by oocyte-secreted factors in primary human cumulus cells. Moreover, our novel findings establish that the combination of GDF9 + BMP15 potently stimulates AMH expression.

scholarly journals Resveratrol Stimulates Cortisol Biosynthesis by Activating SIRT-Dependent Deacetylation of P450scc

Endocrinology ◽  
2012 ◽  
Vol 153 (7) ◽  
pp. 3258-3268 ◽  
Author(s):  
Donghui Li ◽  
Eric B. Dammer ◽  
Marion B. Sewer
Keyword(s):  
Protein Expression ◽  
Fold Increase ◽  
Pyridine Nucleotide ◽  
Nucleotide Metabolism ◽  
Side Chain ◽  
Mitochondrial Enzyme ◽  
Adrenocortical Cells ◽  
Protein Levels ◽  
Chain Cleavage ◽  
Cleavage Enzyme

In the human adrenal cortex, cortisol is synthesized from cholesterol by members of the cytochrome P450 superfamily and hydroxysteroid dehydrogenases. Both the first and last steps of cortisol biosynthesis occur in mitochondria. Based on our previous findings that activation of ACTH signaling changes the ratio of nicotinamide adenine dinucleotide (NAD) phosphate to reduced NAD phosphate in adrenocortical cells, we hypothesized that pyridine nucleotide metabolism may regulate the activity of the mitochondrial NAD+-dependent sirtuin (SIRT) deacetylases. We show that resveratrol increases the protein expression and half-life of P450 side chain cleavage enzyme (P450scc). The effects of resveratrol on P450scc protein levels and acetylation status are dependent on SIRT3 and SIRT5 expression. Stable overexpression of SIRT3 abrogates the cellular content of acetylated P450scc, concomitant with an increase in P450scc protein expression and cortisol secretion. Mutation of K148 and K149 to alanine stabilizes the expression of P450scc and results in a 1.5-fold increase in pregnenolone biosynthesis. Finally, resveratrol also increases the protein expression of P450 11β, another mitochondrial enzyme required for cortisol biosynthesis. Collectively, this study identifies a role for NAD+-dependent SIRT deacetylase activity in regulating the expression of mitochondrial steroidogenic P450.

scholarly journals Determination of CYP450 Expression Levels in the Human Small Intestine by Mass Spectrometry-Based Targeted Proteomics

2021 ◽  
Vol 22 (23) ◽  
pp. 12791
Author(s):  
Alexia Grangeon ◽  
Valérie Clermont ◽  
Azemi Barama ◽  
Fleur Gaudette ◽  
Jacques Turgeon ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Small Intestine ◽  
Protein Expression ◽  
Mrna Levels ◽  
Targeted Proteomics ◽  
Tissue Samples ◽  
Human Organ ◽  
Expression Levels ◽  
Protein Levels ◽  
Human Small Intestine

The human small intestine can be involved in the first-pass metabolism of drugs. Under this condition, members of the CYP450 superfamily are expected to contribute to drug presystemic biotransformation. The aim of this study was to quantify protein expression levels of 16 major CYP450 isoforms in tissue obtained from nine human organ donors in seven subsections of the small intestine, i.e., duodenum (one section, N = 7 tissue samples), jejunum (three subsections (proximal, mid and distal), N = 9 tissue samples) and ileum (three subsections, (proximal, mid and distal), N = 9 tissue samples), using liquid chromatography tandem mass spectrometry (LC-MS/MS) based targeted proteomics. CYP450 absolute protein expression levels were compared to mRNA levels and enzyme activities by using established probe drugs. Proteins corresponding to seven of sixteen potential CYP450 isoforms were detected and quantified in various sections of the small intestine: CYP2C9, CYP2C19, CYP2D6, CYP2J2, CYP3A4, CYP3A5 and CYP4F2. Wide inter-subject variability was observed, especially for CYP2D6. CYP2C9 (p = 0.004) and CYP2C19 (p = 0.005) expression levels decreased along the small intestine. From the duodenum to the ileum, CYP2J2 (p = 0.001) increased, and a trend was observed for CYP3A5 (p = 0.13). CYP3A4 expression was higher in the jejunum than in the ileum (p = 0.03), while CYP4F2 expression was lower in the duodenum compared to the jejunum and the ileum (p = 0.005). CYP450 protein levels were better correlated with specific isoform activities than with mRNA levels. This study provides new data on absolute CYP450 quantification in human small intestine that could improve physiologically based pharmacokinetic models. These data could better inform drug absorption profiles while considering the regional expression of CYP450 isoforms.

GCM2 Silencing in Parathyroid Adenoma is associated with Promoter Hypermethylation and Gain of Methylation on Histone 3

2021 ◽  
Author(s):  
Priyanka Singh ◽  
Sanjay Kumar Bhadada ◽  
Divya Dahiya ◽  
Uma Nahar Saikia ◽  
Ashutosh Kumar Arya ◽  
...  
Keyword(s):  
Protein Expression ◽  
Parathyroid Adenoma ◽  
Dna Methyltransferase ◽  
Promoter Hypermethylation ◽  
Epigenetic Alterations ◽  
Protein Levels ◽  
Histone 3 ◽  
Parathyroid Adenomas ◽  
Mrna And Protein Expression

Abstract Purpose Glial cells missing 2 (GCM2), a zinc finger-transcription factor, is essentially required for the development of parathyroid glands. We sought to identify if the epigenetic alterations in the GCM2 transcription are involved in the pathogenesis of sporadic parathyroid adenoma. In addition, we examined the association between promoter methylation and histone modifications with disease indices. Experimental design mRNA and protein expression of GCM2 were analyzed by RT-qPCR and immunohistochemistry in 33 adenomatous and 10 control parathyroid tissues. DNA methylation and histone methylation/acetylation of GCM2 promoter were measured by bisulfite sequencing and ChIP-qPCR. Additionally, we investigated the role of epigenetic modifications on GCM2 and DNA methyltransferase 1 (DNMT1) expression in PTH-C1 cells by treating with 5-aza 2’deoxycytidine (DAC) and BRD4770 and assessed for GCM2 mRNA and DNMT1 protein levels. Results mRNA and protein expression of GCM2 were lower in sporadic adenomatous than in control parathyroid tissues. This reduction correlated with hypermethylation (P&lt;0.001) and higher H3K9me3 levels in GCM2 promoter (P&lt;0.04) in adenomas. In PTH-C1 cells, DAC treatment resulted in increased GCM2 transcription and decreased DNMT1 protein expression, while cells treated with the BRD4770 showed reduced H3K9me3 levels but a non-significant change in GCM2 transcription. Conclusion These findings suggest the concurrent association of promoter hypermethylation and higher H3K9me3 with the repression of GCM2 expression in parathyroid adenomas. Treatment with DAC restored GCM2 expression in PTH-C1 cells. Our results showed a possible epigenetic landscape in the tumorigenesis of parathyroid adenoma and also that DAC may be promising avenues of research for parathyroid adenoma therapeutics.

Abstract 492: Arachidonic Acid Induces Activation of Platelet PF4 and Par-1 mRNA, Which Is Attenuated by Aspirin

2012 ◽  
Vol 32 (suppl_1) ◽  
Author(s):  
Liang Hu ◽  
Michael A Nardi ◽  
Michael Merolla ◽  
Yajaira Suarez ◽  
Jeffrey Berger
Keyword(s):  
Arachidonic Acid ◽  
Platelet Activation ◽  
Mrna Levels ◽  
Dependent Manner ◽  
Thiazole Orange ◽  
Platelet Activity ◽  
Protein Levels ◽  
Reticulated Platelets ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Arachidonic acid (AA) is converted to thromboxane A2 via the cyclooxygenase pathway; however its exact mechanism of platelet activation is uncertain. Inhibition of this pathway via aspirin highlights the importance of this pathway in decreasing thrombotic events. In the present study, we investigate the effect of AA on platelet activity indicators (leukocyte- and monocyte-platelet aggregation [LPA, MPA] and reticulated platelets [RP]), as well as the expression (mRNA and protein) of platelet markers PF4 and Par-1, previously well established platelet transcripts with quantitative determinations. To this end, whole blood was incubated with AA (150mM) for 30 min at room temperature in the absence or presence of aspirin (1mM) prior to addition of antibodies for platelet activity indicators, and isolating platelets for mRNA and protein expression. LPA and MPA were significantly increased after AA stimulation in a dose dependent manner, and were inhibited by aspirin treatment. AA significantly increased PF4 and Par-1 protein level as determined by flow cytometry and western blot assays. Pretreatment with aspirin also attenuated this increase in protein levels. Surprisingly, AA stimulation significantly increased thiazole orange staining (a measure of nucleic acids), another marker of increased platelet activity. Importantly, these results suggest that AA-mediated platelet activation produced an overall increase in platelet total RNA content. To confirm these findings, we analyzed the mRNA expression of PF4 and Par-1 by quantitative real time PCR from platelets treated with AA. Interestingly, AA significantly up-regulated the platelet mRNA transcripts of PF4 and Par-1 by 40% to 60%, and pretreatment with aspirin completely attenuated this effect supporting the specificity of the AA effect on platelet RNA. Altogether, these data suggest that platelet mRNA is affected by AA stimulation, which is attenuated by pretreatment with aspirin. However, the mechanisms responsible for the increased mRNA levels and expression of PF4 and Par-1 (processing of pre-RNA to mRNA) require further investigation. Importantly, our findings provide novel insight regarding platelet activation and a better understanding of mediators in the processes of thrombosis and hemostasis.

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