scholarly journals Cloning and Expression Analysis of Two Kdm Lysine Demethylases in the Testes of Mature Yaks and Their Sterile Hybrids

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 521
Author(s):  
Zhenhua Shen ◽  
Lin Huang ◽  
Suyu Jin ◽  
Yucai Zheng
Keyword(s):  
Amino Acids ◽  
Protein Expression ◽  
Male Sterility ◽  
Histone Methylation ◽  
Real Time Pcr ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Coding Sequences ◽  
Mrna And Protein Expression ◽  
Sterile Hybrids

The objective of this study was to explore the molecular mechanism for male sterility of yak hybrids based on two demethylases. Total RNA was extracted from the testes of adult yaks (n = 10) and yak hybrids (cattle–yaks, n = 10). The coding sequences (CDS) of two lysine demethylases (KDMs), KDM1A and KDM4B, were cloned by RT-PCR. The levels of KDM1A and KDM4B in yaks and cattle–yaks testes were detected using Real-time PCR and Western blotting for mRNA and protein, respectively. In addition, the histone methylation modifications of H3K36me3 and H3K27me3 were compared between testes of yaks and cattle–yaks using ELISA. The CDS of KDM1A and KDM4B were obtained from yak testes. The results showed that the CDS of KDM1A exhibited two variants: variant 1 has a CDS of 2622 bp, encoding 873 amino acids, while variant 2 has a CDS of 2562 bp, encoding 853 amino acids. The CDS of the KDM4B gene was 3351 bp in length, encoding 1116 amino acids. The mRNA and protein expression of KDM1A and KDM4B, as well as the level of H3K36me3, were dramatically decreased in the testes of cattle–yaks compared with yaks. The present results suggest that the male sterility of cattle–yaks might be associated with reduced histone methylation modifications.

scholarly journals Hypoxia increases KIAA1199/CEMIP expression and enhances cell migration in pancreatic cancer

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Takuya Oba ◽  
Norihiro Sato ◽  
Yasuhiro Adachi ◽  
Takao Amaike ◽  
Yuzan Kudo ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Expression ◽  
Hypoxia Inducible Factor ◽  
Ductal Adenocarcinoma ◽  
Rt Pcr ◽  
Transwell Migration Assay ◽  
Migration Assay ◽  
Hypoxic Conditions ◽  
Mrna And Protein Expression ◽  
Interfering Rna

AbstractPancreatic ductal adenocarcinoma (PDAC) is characterised by dense desmoplasia and hypoxic microenvironment. Our previous reports demonstrated that hyaluronan (HA), especially low-molecular-weight HA, provides a favourable microenvironment for PDAC progression. However, the effect of hypoxia on HA metabolism remains unknown. Using quantitative real-time RT-PCR and western blot analysis, we analysed the changes in the expression of HA-synthesizing enzymes (HAS2 and HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic conditions. Hypoxia increased the mRNA and protein expression of KIAA1199, whereas it decreased HYAL1 expression. The expression of HAS3 was increased and HAS2 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined using a transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced the migratory ability of PDAC cells, which was inhibited by KIAA1199 knockdown. We also used immunohistochemistry to analyse the protein expression of hypoxia inducible factor (HIF) 1α and KIAA1199 in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

5-HT2B receptors are expressed on astrocytes from brain and in culture and are a chronic target for all five conventional ‘serotonin-specific reuptake inhibitors’

2010 ◽  
Vol 6 (2) ◽  
pp. 113-125 ◽  
Author(s):  
Shiquen Zhang ◽  
Baoman Li ◽  
Ditte Lovatt ◽  
Junnan Xu ◽  
Dan Song ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cell Sorting ◽  
Cultured Cells ◽  
Primary Cultures ◽  
Rt Pcr ◽  
Chain Reaction ◽  
Calcium Dependent ◽  
Mrna And Protein Expression ◽  
Polymerase Chain ◽  
Well Differentiated

In well-differentiated primary cultures of mouse astrocytes, which express no serotonin transporter (SERT), the ‘serotonin-specific reuptake inhibitor’ (SSRI) fluoxetine leads acutely to 5-HT2B receptor-mediated, transactivation-dependent phosphorylation of extracellular regulated kinases 1/2 (ERK1/2) with an EC50 of ~5 μM, and chronically to ERK1/2 phosphorylation-dependent upregulation of mRNA and protein expression of calcium-dependent phospholipase A2 (cPLA2) with ten-fold higher affinity. This affinity is high enough that fluoxetine given therapeutically may activate astrocytic 5-HT2B receptors (Li et al., 2008, 2009). We now confirm the expression of 5-HT2B receptors in astrocytes freshly dissociated from mouse brain and isolated by fluorescence-activated cell sorting (FACS) and investigate in cultured cells if the effects of fluoxetine are shared by all five conventional SSRIs with sufficiently high affinity to be relevant for mechanism(s) of action of SSRIs. Phosphorylated and total ERK1/2 and mRNA and protein expression of cPLA2a were determined by Western blot and reverse transcription polymerase chain reaction (RT-PCR). Paroxetine, which differs widely from fluoxetine in affinity for SERT and for another 5-HT2 receptor, the 5-HT2C receptor, acted acutely and chronically like fluoxetine. One micromolar of paroxetine, fluvoxamine or sertraline increased cPLA2a expression during chronic treatment; citalopram had a similar effect at 0.1–0.5 μM; these are therapeutically relevant concentrations.

scholarly journals FGF21 Ameliorates Hepatic Fibrosis by Multiple Mechanisms

2021 ◽  
Author(s):  
Fanrui Meng ◽  
Mir Hassan Khoso ◽  
Kai Kang ◽  
Qi He ◽  
Yukai Cao ◽  
...  
Keyword(s):  
Protein Expression ◽  
Western Blot ◽  
Hepatic Fibrosis ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Cell Model ◽  
Dependent Manner ◽  
Rt Pcr ◽  
Mrna And Protein Expression ◽  
Dose Dependent

Abstract Previous study reports that FGF21 could ameliorate hepatic fibrosis, but its mechanisms have not been fully investigated. In this study, three models were used to investigate the mechanism by which FGF21 alleviates liver fibrosis. CCL4 and DMN were respectively used to induce hepatic fibrosis animal models. Our results demonstrated that liver index and liver function were deteriorated in both models. HE and Masson’s staining showed that the damaged tissue architectonics were observed in the mice of both models. Treatment with FGF21 significantly ameliorated these changes. ELISA analysis showed that the serum levels of IL-1β, IL-6 and TNF-α were significantly elevated in both models. However, administration of FGF21 significantly reduced these inflammatory cytokines. RT-PCR and Western blot analysis showed that mRNA and protein expression of collagenI, α-SMA and TGF-β were significantly decreased by treatment with FGF21. PDGF-BB stimulant was used to establish the experimental cell model in HSCs. RT-PCR and Western blot analysis demonstrated that the expression of collagenI and α-SMA were significantly upregulated by this stimulant in model group. Interestingly, our results showed that mRNA and protein expression of leptin were also significantly induced in PDGF-BB treated HSCs. Administration of FGF21 could significantly reduce leptin expression in a dose dependent manner and these effects were reversed in siRNA (against β-klotho) transfected HSCs. Furthermore, the leptin signaling pathways related protein p-ERK/t-ERK, p-STAT3/STAT3 and TGF-β were significantly downregulated by FGF21 treatment in a dose dependent manner. The expression of SOCS3 and Nrf-2 were enhanced by treatment with FGF21. The underlying mechanism may be that FGF21 regulates leptin-STAT3 axis via Nrf-2 and SOCS3 pathway in activated HSCs.

Regulation by glucocorticoids and osmolality of expression of ROMK (Kir 1.1), the apical K channel of thick ascending limb

2003 ◽  
Vol 284 (5) ◽  
pp. F977-F986 ◽  
Author(s):  
Morgan Gallazzini ◽  
Amel Attmane-Elakeb ◽  
David B. Mount ◽  
Steven C. Hebert ◽  
Maurice Bichara
Keyword(s):  
Protein Expression ◽  
In Vivo Studies ◽  
K Channel ◽  
Thick Ascending Limb ◽  
Rt Pcr ◽  
Immunoblotting Analysis ◽  
Medullary Thick Ascending Limb ◽  
Nacl Concentration ◽  
Mrna And Protein Expression

Mechanisms of regulation of ROMK channel mRNA and protein expression in medullary thick ascending limb (MTAL) were assessed in rat MTAL fragments incubated for 7 h. ROMK mRNA was quantified by quantitative RT-PCR and ROMK protein by immunoblotting analysis of crude membranes. Medium hyperosmolality (450 mosmol/kgH2O; NaCl plus urea added to isoosmotic medium) increased ROMK mRNA ( P < 0.04) and protein ( P < 0.006), and 10 nM dexamethasone also increased ROMK mRNA ( P < 0.02). Hyperosmolality and dexamethasone had no additive effects on ROMK mRNA. NaCl alone, but not urea or mannitol, reproduced the hyperosmolality effect on ROMK mRNA. 1-Deamino-(8-d-arginine) vasopressin (1 nM) or 0.5 mM 8-bromo-cAMP had no effect per se on ROMK mRNA and protein. However, 8-bromo-cAMP abolished the stimulatory effect of dexamethasone on ROMK mRNA in the isoosmotic but not in the hyperosmotic medium ( P < 0.004). In in vivo studies, the abundance of ROMK protein and mRNA increased in adrenalectomized (ADX) rats infused with dexamethasone compared with ADX rats ( P < 0.02). These results establish glucocorticoids and medium NaCl concentration as direct regulators of MTAL ROMK mRNA and protein expression, which may be modulated by cAMP-dependent factors.

scholarly journals Histological Analysis, Bioinformatics Profile, and Expression of Methylenetetrahydrofolate Reductase (MTHFR) in Bovine Testes

Animals ◽  
2020 ◽  
Vol 10 (10) ◽  
pp. 1731
Author(s):  
Seth Afedo ◽  
Yan Cui ◽  
Sijiu Yu ◽  
Bo Liao ◽  
Zihan Zhao ◽  
...  
Keyword(s):  
Amino Acids ◽  
Protein Expression ◽  
Methylenetetrahydrofolate Reductase ◽  
Protein Structures ◽  
Zebu Cattle ◽  
Reading Frame ◽  
Physico Chemical ◽  
Yellow Cattle ◽  
Mrna And Protein Expression ◽  
Sequence Encoding

Methylenetetrahydrofolate reductase (MTHFR), an enzyme expressed in mammalian testes, exerts a direct effect on spermatogenesis; however, its protein characteristics in bovine testes remain unknown. Here, we analysed bovine testicular structure, MTHFR bioinformatics profile, mRNA, and protein expression characteristics in yellow-cattle (y-c) and yak testis using histological procedures, bioinformatics analysis, qRT-PCR, and western blot. Testes from 13 bovines, ≤2 years juvenile (y-c, n = 3; yak, n = 3) and ≥3 years adult (y-c, n = 3; yak, n = 4) were collected and analysed. Anatomical characteristics of testis in y-c and yak were similar except the weight or size for which that of y-c was significantly higher or greater than yak. In y-c, an open reading frame (ORF) for 2600 nucleotides sequence, encoding 655 amino acids showed high homology with zebu cattle (99.51%) and wild yak (98.68%). Secondary and 3D protein structures were similar to that of humans with differences in the number of nucleotides, amino acids, and some physico-chemical characteristics. MTHFR mRNA expression in y-c and yak were significantly higher in adult testes compared with juvenile ones. However, its protein expression was higher, but not statistically significant, in adult y-c and yak compared to the juvenile ones. The highlights and inferences of these and other findings are discussed.

scholarly journals Hypoxia Increases KIAA1199/CEMIP Expression and Enhances Cell Migration in Pancreatic Cancer

2021 ◽  
Author(s):  
Takuya Oba ◽  
Norihiro Sato ◽  
Yasuhiro Adachi ◽  
Takao Amaike ◽  
Yuzan Kudo ◽  
...  
Keyword(s):  
Cell Migration ◽  
Protein Expression ◽  
Hypoxia Inducible Factor ◽  
Hypoxic Condition ◽  
Ductal Adenocarcinoma ◽  
Rt Pcr ◽  
Transwell Migration Assay ◽  
Migration Assay ◽  
Mrna And Protein Expression ◽  
Interfering Rna

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense desmoplasia and hypoxic microenvironment. We previously demonstrated that hyaluronan (HA), especially low-molecular weight HA, provides a favorable microenvironment for progression of PDAC. However, the effect of hypoxia on HA metabolism is unknown. Using quantitative real-time RT-PCR and Western blot analysis, we analyzed changes in expression of HA-synthesizing enzymes (HAS2, HAS3) and HA-degrading enzymes (HYAL1, KIAA1199/CEMIP) in PDAC cell lines under hypoxic condition. Hypoxia increased mRNA and protein expression of KIAA1199, whereas it decreased expression of HYAL1. Expression of HAS2 and HAS3 remained unchanged in response to hypoxia. The effect of KIAA1199 on hypoxia-induced cell migration was determined by transwell migration assay and small-interfering RNA (siRNA). Hypoxia enhanced migratory ability of PDAC cells, which was inhibited by knockdown of KIAA1199 expression. We also used immunohistochemistry to analyze Hypoxia Inducible Factor (HIF) 1α and KIAA1199 protein expression in PDAC tissues. There was a significant immunohistochemically positive correlation between KIAA1199 and HIF1α. These findings suggest that hypoxia-induced KIAA1199 expression may contribute to enhanced motility in PDAC.

Type 2 diabetes: increased expression and contribution of IKCa channels to vasodilation in small mesenteric arteries of ZDF rats

2014 ◽  
Vol 307 (8) ◽  
pp. H1093-H1102 ◽  
Author(s):  
Christian Schach ◽  
Markus Resch ◽  
Peter M. Schmid ◽  
Guenter A. Riegger ◽  
Dierk H. Endemann
Keyword(s):  
Type 2 Diabetes ◽  
Protein Expression ◽  
Experimental Models ◽  
Diabetic Rats ◽  
Mesenteric Arteries ◽  
Rt Pcr ◽  
Mrna And Protein Expression ◽  
Zdf Rats ◽  
Endothelium Dependent Relaxation

Impaired endothelial function, which is dysregulated in diabetes, also precedes hypertension. We hypothesized that in Type 2 diabetes, the impaired endothelium-dependent relaxation is due to a loss of endothelium-derived hyperpolarization (EDH) that is regulated by impaired ion channel function. Zucker diabetic fatty (ZDF), Zucker heterozygote, and homozygote lean control rats were used as the experimental models in our study. Third-order mesenteric arteries were dissected and mounted on a pressure myograph; mRNA was quantified by RT-PCR and channel proteins by Western blotting. Under nitric oxide (NO) synthase and cyclooxygenase inhibition, endothelial stimulation with ACh fully relaxes control but not diabetic arteries. In contrast, when small-conductance calcium-activated potassium (KCa) channels and intermediate- and large-conductance KCa (I/BKCa) are inhibited with apamin and charybdotoxin, NO is able to compensate for ACh-induced relaxation in control but not in diabetic vessels. After replacement of charybdotoxin with 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34; IKCa inhibitor), ACh-induced relaxation in diabetic animals is attenuated. Specific inhibition with TRAM-34 or charybdotoxin attenuates ACh relaxation in diabetes. Stimulation with 1-ethyl-2-benzimidazolinone (IKCa activator) shows a reduced relaxation in diabetes. Activation of BKCa with 1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl)phenyl]-5-(trifluoromethyl)-2H-benzimidazol-2-one NS619 leads to similar relaxations of control and diabetic arteries. RT-PCR and Western blot analysis demonstrate elevated mRNA and protein expression levels of IKCa in diabetes. Our results suggest that the compensatory effect of NO and EDH-associated, endothelium-dependent relaxation is reduced in ZDF rats. Specific blockade of IKCa with TRAM-34 reduces NO and EDH-type relaxation in diabetic rats, indicating an elevated contribution of IKCa in diabetic small mesenteric artery relaxation. This finding correlates with increased IKCa mRNA and protein expression in this vessel.

scholarly journals Effect of MT3 on Retinal and Choroidal TGF-β2 and HAS2 Expressions in Form Deprivation Myopia of Guinea Pig

2017 ◽  
Vol 2017 ◽  
pp. 1-9
Author(s):  
Tao Li ◽  
Xiaodong Zhou ◽  
Bing Li ◽  
Bo Jiang
Keyword(s):  
Protein Expression ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Protein Expression Level ◽  
Tissue Concentrations ◽  
Treatment Groups ◽  
Protein Expressions ◽  
Mrna And Protein Expression ◽  
Dose Dependent ◽  
Dose Dependent Effect

Purpose. To confirm its dose-dependent effect on form deprivation myopia and evaluate the effect of MT3 at different tissue concentrations on changes in mRNA and protein expression for TGF-β2 and HAS2. Methods. MT3 was intravitreally injected into deprived eyes at two-day intervals. Refraction was measured by streak retinoscopy after cycloplegia. The axial dimensions were measured by A-scan ultrasound. The quantitative RT-PCR and Western blot were used to detect the changes of TGF-β2 and HAS2 expressions in the retina and choroid of guinea pigs. Results. MT3 treatment produced a significant dose-dependent reduction in relative myopia compared to FD group (both p<0.001). There were statistically significant increases in retinal and choroidal mRNA levels for both TGF-β2 and HAS2 after injections of 10 μM of MT3, when compared to the FD group. There were no significant differences in retinal and choroidal TGF-β2 protein expression levels between the MT3 treatment groups and FD group (all p>0.05). The injections of 10 μM of MT3 caused a marked decrease in retinal HAS2 protein expression level, when compared to the FD group (p=0.001). Conclusion. MT3 can inhibit form deprivation myopia, and MT3 treatment can result in changes of retinal and choroidal TGF-β2 and HAS2 mRNA and protein expressions.

Cloning and Expression Analysis of ALDH Conserved cDNA Region from Fraxinus velutina

2011 ◽  
Vol 282-283 ◽  
pp. 616-620
Author(s):  
Fan Suo Zeng ◽  
Hui Wei Zhang ◽  
Ya Guang Zhan ◽  
Ying Xin
Keyword(s):  
Amino Acids ◽  
Aldehyde Dehydrogenase ◽  
Aromatic Aldehydes ◽  
Nacl Stress ◽  
Cloning And Expression ◽  
Rt Pcr ◽  
Conserved Sequence ◽  
Fraxinus Velutina ◽  
Dehydrogenase Gene ◽  
Wide Range

Aldehyde dehydrogenases (ALDHs) represent a protein superfamily of NAD(P)+-dependent enzymes that oxidize a wide range of endogenous and exogenous aliphatic and aromatic aldehydes. The conserved sequence of aldehyde dehydrogenase gene (ALDH) was amplified fromFraxinus velutinaby RT-PCR. The results demonstrated that the conserved sequence was 718 bp in length, which was deduced coding 229 amino acids. Homology analysis showed that the deduced amino acids sequence ofFvALDHshared 56.9%%-73% identity with aldehyde dehydrogenase from other plants. We examined the expression pattern of theFvALDHgene in leaf treated with NaCl stress for defferent time using semi-quantitative RT-PCR. The results showed that the expression ofFvALDHwas increased after the first decreased, and then in a steady state with the time went on.

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