scholarly journals Aquaporin Expression in the Fetal Porcine Urinary Tract Changes During Gestation

2018 ◽  
pp. 283-292 ◽  
Author(s):  
L. K. JAKOBSEN ◽  
K. F. TRELBORG ◽  
P. S. KINGO ◽  
S. HØYER ◽  
K.-E. ANDERSSON ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Mrna Expression ◽  
Western Blotting ◽  
Gestational Age ◽  
Renal Pelvis ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Protein Levels ◽  
Water Handling ◽  
Aqp1 Expression

The expression of aquaporins (AQPs) in the fetal porcine urinary tract and its relation to gestational age has not been established. Tissue samples from the renal pelvis, ureter, bladder and urethra were obtained from porcine fetuses. Samples were examined by RT-PCR (AQPs 1-11), QPCR (AQPs positive on RT-PCR), and immunohistochemistry. Bladder samples were additionally examined by Western blotting. RNA was extracted from 76 tissue samples obtained from 19 fetuses. Gestational age was 60 (n=11) or 100 days (n=8). PCR showed that AQP1, 3, 9 and 11 mRNA was expressed in all locations. The expression of AQP3 increased significantly at all four locations with gestational age, whereas AQP11 significantly decreased. AQP1 expression increased in the ureter, bladder and urethra. AQP9 mRNA expression increased in the urethra and bladder, but decreased in the ureter. AQP5 was expressed only in the urethra. Immunohistochemistry showed AQP1 staining in sub-urothelial vessels at all locations. Western blotting analysis confirmed increased AQP1 protein levels in bladder samples during gestation. Expression levels of AQP1, 3, 5, 9 and 11 in the urinary tract change during gestation, and further studies are needed to provide insights into normal and pathophysiological water handling mechanisms in the fetus.

scholarly journals Expression of ACE2, the SARS-CoV-2 receptor, in lung tissue of patients with type 2 diabetes

2020 ◽  
Author(s):  
Ada Admin ◽  
Sara RA Wijnant ◽  
Merel Jacobs ◽  
Hannelore P Van Eeckhoutte ◽  
Bruno Lapauw ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Lung Tissue ◽  
Bronchial Epithelium ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Protein Levels ◽  
Patients With Diabetes ◽  
Alveolar Tissue ◽  
Control Samples

Increased expression of pulmonary ACE2, the SARS-CoV-2 receptor, could contribute to increased infectivity of COVID-19 in subjects with diabetes, but ACE2 expression has not been studied in lung tissue of subjects with diabetes. We therefore studied ACE2 mRNA and protein expression in lung tissue samples of patients with and without diabetes that were collected between 2002 and 2020 from patients undergoing lobectomy for lung tumors. For RT-PCR analyses, samples from 15 subjects with diabetes were compared to 91 randomly chosen control samples. For immunohistochemical staining, samples from 26 subjects with diabetes were compared to 66 randomly chosen control samples. mRNA expression of ACE2 was measured by quantitative RT-PCR. Protein levels of ACE2 were visualized by immunohistochemistry on paraffin-embedded lung tissue samples and quantified in alveolar and bronchial epithelium. Pulmonary ACE2 mRNA expression was not different between subjects with or without diabetes. In contrast, protein levels of ACE2 were significantly increased in both alveolar tissue and bronchial epithelium of patients with diabetes as compared with control subjects, independent of smoking, COPD, BMI, RAAS-inhibitor use and other potential confounders. To conclude, we show increased bronchial and alveolar ACE2 protein expression in patients with diabetes. Further research is needed to elucidate whether up-regulation of ACE2 expression in airways and lungs has consequences on infectivity and clinical outcomes of COVID-19.

scholarly journals Expression of ACE2, the SARS-CoV-2 receptor, in lung tissue of patients with type 2 diabetes

2020 ◽  
Author(s):  
Ada Admin ◽  
Sara RA Wijnant ◽  
Merel Jacobs ◽  
Hannelore P Van Eeckhoutte ◽  
Bruno Lapauw ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Lung Tissue ◽  
Bronchial Epithelium ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Protein Levels ◽  
Patients With Diabetes ◽  
Alveolar Tissue ◽  
Control Samples

Increased expression of pulmonary ACE2, the SARS-CoV-2 receptor, could contribute to increased infectivity of COVID-19 in subjects with diabetes, but ACE2 expression has not been studied in lung tissue of subjects with diabetes. We therefore studied ACE2 mRNA and protein expression in lung tissue samples of patients with and without diabetes that were collected between 2002 and 2020 from patients undergoing lobectomy for lung tumors. For RT-PCR analyses, samples from 15 subjects with diabetes were compared to 91 randomly chosen control samples. For immunohistochemical staining, samples from 26 subjects with diabetes were compared to 66 randomly chosen control samples. mRNA expression of ACE2 was measured by quantitative RT-PCR. Protein levels of ACE2 were visualized by immunohistochemistry on paraffin-embedded lung tissue samples and quantified in alveolar and bronchial epithelium. Pulmonary ACE2 mRNA expression was not different between subjects with or without diabetes. In contrast, protein levels of ACE2 were significantly increased in both alveolar tissue and bronchial epithelium of patients with diabetes as compared with control subjects, independent of smoking, COPD, BMI, RAAS-inhibitor use and other potential confounders. To conclude, we show increased bronchial and alveolar ACE2 protein expression in patients with diabetes. Further research is needed to elucidate whether up-regulation of ACE2 expression in airways and lungs has consequences on infectivity and clinical outcomes of COVID-19.

scholarly journals Ultrasound microbubble-mediated CRISPR/Cas9 knockout of C-erbB-2 in HEC-1A cells

2019 ◽  
Vol 47 (5) ◽  
pp. 2199-2206 ◽  
Author(s):  
Junhong Cai ◽  
Sizhe Huang ◽  
Yuping Yi ◽  
Shan Bao
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Western Blotting ◽  
Growth Factor Receptor ◽  
Negative Control ◽  
Rt Pcr ◽  
Control Groups ◽  
Transfected Cells ◽  
Significant Difference ◽  
Ultrasound Microbubbles

Objective Epidermal growth factor receptor 2 (C-erbB-2) is one of the most frequently mutated oncogenes in human tumors. We aimed to evaluate the knockout efficiency of clustered regularly interspaced short palindromic repeat (CRISPR) technology using ultrasound microbubble transfection to target C-erbB-2 in human endometrial cancer (HEC)-1A cells. Methods Three single guide RNAs (sgRNAs) targeting C-erbB-2 were designed and used to construct CRISPR/CRISPR-associated (Cas)9-C-erbB-2 plasmids. The constructed plasmids were transfected into HEC-1A cells using ultrasound microbubbles. C-erbB-2 knockout cloned cells were identified by green fluorescence. C-erbB-2 mRNA and protein expression was measured by reverse transcription (RT)-PCR and western blotting, respectively. Results RT-PCR showed that C-erbB-2 mRNA expression was significantly lower in sgRNA1-transfected cells (0.57 ± 0.06) than in blank (1.00 ± 0.09) and negative-control groups (1.02 ± 0.12). Western blotting revealed C-erbB-2 protein expression to be significantly lower in sgRNA1-transfected cells (0.269 ± 0.033) than in blank (0.495 ± 0.059) and negative-control groups (1.243 ± 0.281). However, there was no significant difference in C-erbB-2 protein and mRNA expression in sgRNA2- and sgRNA3-transfected cells compared with controls. Conclusion Ultrasound microbubbles can mediate plasmid transfer into HEC-1A cells to interfere with gene expression and knockout C-erbB-2.

255 GENE EXPRESSION OF Cox 5a, 5b, AND 6b1 AND THEIR ROLES IN PRE-IMPLANTATION OF MOUSE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 235
Author(s):  
S.-E. Lee ◽  
X.-Y. Li ◽  
X.-S. Cui ◽  
N.-H. Kim
Keyword(s):  
Rna Interference ◽  
Mrna Expression ◽  
Real Time ◽  
Oocyte Maturation ◽  
Blastocyst Stage ◽  
Mrna Levels ◽  
Rt Pcr ◽  
Target Mrna ◽  
Protein Levels

Despite clear evidence of regulation of mitochondrial respiration by nuclear encoded genes, cytochrome oxidase (Cox), little information is available on their expression and functional roles during early embryonic development. To examine the role of Cox in oocyte maturation and embryogenesis, we first characterized mRNA and protein levels of nuclear encoded genes, Cox 5a, 5b, and 6b1, in mouse oocytes and during early embryogenesis, using real-time RT-PCR and immunocytochemistry. We then examined the possible role of these genes in oocyte maturation and pre-implantation development using RNA interference analysis. The relative abundances of Cox 5a, 5b, and 6b1 transcripts was measured by real time RT-PCR. After normalization by comparison to histone H2a mRNA levels, the mRNA expression of Cox 5a, 5b, and 6b1 were found to be considerable in mature oocytes and zygotes, but reduced slightly in 2-cell embryos. From the 2-cell to the blastocyst stage, mRNA expression is dependent on the number of blastomeres, as expression increases only gradually with development. Immunocytochemical studies revealed that Cox 5a, 5b, and 6b1 proteins were expressed in all blastomeres of the blastocyst. Injection of Cox 5a, 5b, or 6b1 siRNA into GV stage oocytes decreased expression of the target mRNA specifically, while not affecting the expression of mRNAs for the other subunits in mature oocytes. Similarly, each siRNA injection into zygotes specifically reduced target mRNA expression at the 2-cell, morula and blastocyst stages (P < 0.05). Silencing of mRNA expression by RNA interference (siRNA) did not inhibit oocyte maturation or developmental events up to the morula and blastocyst stages. The expression level of mtDNA9, as well as overall levels of mitochondrial mRNAs, was not different following injection of siRNA for Cox 5a, 5b, or 6b1. However, it is evident that the number of mitochondria in siRNA treated blastocysts was greatly reduced, and they appeared to be morphologically abnormal. Significantly higher apoptosis and lower cell numbers were observed in siRNA treated blastocysts. Real time RT PCR revealed that silencing of Cox 5a, 5b, and 6b1 decreased mRNA and protein levels of E-cadherin. These results suggest that the Cox subunits, Cox 5a, 5b, and 6b1, play an important role in mitochondrial function during pre-implantation development. This work was funded by a grant from the National Research Laboratory Program in Korea.

scholarly journals Expression Profile of IL-35 mRNA in Gingiva of Chronic Periodontitis and Aggressive Periodontitis Patients: A Semiquantitative RT-PCR Study

2013 ◽  
Vol 35 ◽  
pp. 819-823 ◽  
Author(s):  
Nagaraj B. Kalburgi ◽  
Akshay Muley ◽  
B. M. Shivaprasad ◽  
Arati C. Koregol
Keyword(s):  
Mrna Expression ◽  
Chronic Periodontitis ◽  
T Regulatory Cells ◽  
Periodontal Diseases ◽  
Aggressive Periodontitis ◽  
Gingival Tissue ◽  
Regulatory Cells ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Polymerase Chain

Background. Proinflammatory and anti-inflammatory cytokines play a key role in the pathogenesis of periodontal diseases. Secretion of bioactive IL-35 has been described by T regulatory cells () and is required for their maximal suppressive activity. are involved in the modulation of local immune response in chronic periodontitis patients.Objective. Hence, the present study was aimed to investigate the expression of IL-35 mRNA in chronic periodontitis and aggressive periodontitis patients.Materials and Methods. The present study was carried out in 60 subjects, which included 20 chronic periodontitis patients, 20 aggressive periodontitis patients, and 20 periodontally healthy controls. IL-35 mRNA expression in gingival tissue samples of all subjects was semiquantitatively analyzed using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR).Results. The present study demonstrated the expression of IL-35 mRNA in gingival tissues of all the three groups. IL-35 mRNA expression was highest in chronic periodontitis subjects () as compared to the aggressive periodontitis group () and least seen in healthy patients ().Conclusion. The increased expression of IL-35 in chronic and aggressive periodontitis suggests its possible role in pathogenesis of periodontitis. Future studies done on large samples with intervention will strengthen our result.

scholarly journals Effect of Ligustrazine on Endometrium Injury of Thin Endometrium Rats

2019 ◽  
Vol 2019 ◽  
pp. 1-7 ◽  
Author(s):  
Qing Ye ◽  
Yu Zhang ◽  
Jiulu Fu ◽  
Yi Zou ◽  
Wei Zhao ◽  
...  
Keyword(s):  
Western Blotting ◽  
Uterine Cavity ◽  
Control Group ◽  
Mrna Levels ◽  
Histopathological Changes ◽  
Model Group ◽  
Rt Pcr ◽  
Thin Endometrium ◽  
Protein Levels ◽  
Hematoxylin Eosin

The purpose of this experiment is to establish a rat model of thin endometrium and to explore the effect of ligustrazine on the thin endometrium of rats. The thin endometrium model was made by using infusing absolute ethyl alcohol into the uterine cavity. The thickness of endometrium was measured. Hematoxylin-Eosin (HE) staining was used to observe the histopathological changes of endometrium. The mRNA levels of VEGF, VEGFR-2, PI3K, and AKT were detected by RT-PCR. Western blotting was used to detect the levels of VEGF, VEGFR-2, PI3K, and AKT in endometrial tissue. The thickness of endometrium in the model group was significantly thinner than that in the control group. Compared with the model group, the thickness of endometrium in ligustrazine group was increased. HE staining shown that ligustrazine restored the histopathological changes of endometrium. RT-PCR and Western Blotting results showed that the mRNA and protein levels of VEGF, VEGFR-2, PI3K, and AKT in the model group were significantly decreased compared with the control group, while ligustrazine restored the changes. Ligustrazine can improve the morphology of endometrium, can promote the growth of endometrium, and has obvious therapeutic effect. Its mechanism is related to the activation of PI3K/Akt signaling pathway through upregulation of VEGF and VEGFR-2 expression to induce the repair of thin endometrium in rats.

scholarly journals The expression of endothelin type A and B receptors in the lateral wall of the mouse cochlea

2007 ◽  
Vol 12 (4) ◽  
Author(s):  
Yan Luo ◽  
Yuedi Tang ◽  
Qingjie Xia ◽  
Jin Liu
Keyword(s):  
Mrna Expression ◽  
Real Time ◽  
Lateral Wall ◽  
Type A ◽  
Mouse Tissue ◽  
Receptor Subtypes ◽  
Biological Functions ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Polymerase Chain

AbstractEndothelin (ET), originally characterized as a vasoconstrictive peptide, has been found to have many different biological functions, including acting as a local hormonal regulator of pressure, fluid, ions and neurotransmitters in the inner ear. The objective of this study was to examine and quantify the mRNA expression of the endothelin type A and B receptors (ETAR and ETBR) in the strial vascularies (StV) and non-strial tissues (NSt) of the cochlear lateral wall using the real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) technique. The mouse tissue samples were harvested and RNA was extracted. RT was performed to obtain cDNA, and then the mRNA expression of each gene was measured via real-time PCR. We found that both receptor subtypes were expressed in the cochlear lateral wall, with a predominance of ETAR over ETBR. We showed that the mRNA expression of the two receptor subtypes was higher in the StV with a 1.8 times higher level of ETAR and an 8.1 times higher level of ETBR mRNAs than in the adjacent NSt of the lateral wall tissue. This study shows the existence and the quantity of ET receptor subtypes in the StV and NSt of the mouse cochlea. Our results suggest that an endothelin-mediated response via two different receptors, ETAR and ETBR, may play an important role in the physiological functions of the cochlear lateral wall by maintaining the homeostatic environment of the cochlea.

scholarly journals Glucocorticoid receptor expression and glucocorticoid therapeutic effect in nasal polyps

2010 ◽  
Vol 33 (3) ◽  
pp. 181 ◽  
Author(s):  
Peng Li ◽  
Yuan Li ◽  
Yong-Qi Li ◽  
Qin-Tai Yang ◽  
Ge-Hua Zhang
Keyword(s):  
Glucocorticoid Receptor ◽  
Mrna Expression ◽  
Therapeutic Effect ◽  
Nasal Mucosa ◽  
Nasal Polyps ◽  
Receptor Expression ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Polymerase Chain ◽  
Sensitive Group

Purpose: To investigate the expression and quantity of glucocorticoid receptor-α and -β in polyp tissues taken from the patients treated were subsequently treated with topical glucocorticoid (GC). Methods: Eighty patients with nasal polyps were initially enrolled in the study. All polyp specimens were obtained prior to treatment. Patients then received daily topical GC spray treatment for one month. Polyp specimens were tested for glucocorticoid receptor (GR) GR-α and GR-β mRNA expression using fluorescent quantitative-reverse transcription-polymerase chain reaction (FQ-RT-PCR). Thirty healthy nasal mucosa tissue samples were tested at the same time. Results: Forty patients finished the study and were divided into two groups: GC-sensitive (n=26) and GC-insensitive (n=14), according to treatment results. GR-β mRNA expression in the nasal polyp tissues of the GC-insensitive group (5.72±0.58×102 copies/μg) was higher than that in the GC-sensitive group (4.82±0.28×102 copies/μg, P < 0.05) and in the normal nasal mucosa group (4.44±0.35×102 copies/μg, P < 0.01). There was also a difference in the relative expression of GR-α and GR-β between the GC-sensitive group (GR-α/GR-β= 829.42±67.36) and the GC-insensitive group (535.7±89) (P < 0.01). Conclusion: GR-β mRNA was highly expressed in patients with nasal polyps. Down- regulation of GR-α mRNA suggests the existence of glucocorticoid insensitivity. Expression of GR-β may plays an important role in the evaluation of the glucocorticoid therapeutic effect in patients with nasal polyps.

scholarly journals The Presence and Distribution of TRPM7 in the Canine Mammary Glands

Animals ◽  
2020 ◽  
Vol 10 (3) ◽  
pp. 466
Author(s):  
Sungin Lee ◽  
Seulji Lee ◽  
Aeri Lee ◽  
Hun Ju Sim ◽  
Geon A. Kim ◽  
...  
Keyword(s):  
Mammary Gland ◽  
Western Blotting ◽  
Transient Receptor Potential ◽  
Expression Patterns ◽  
Mammary Glands ◽  
Normal Mammary Gland ◽  
Rt Pcr ◽  
Tissue Samples ◽  
Transient Receptor Potential Melastatin ◽  
Canine Mammary Gland

The transient receptor potential melastatin-subfamily member 7 (TRPM7) cation channel is a bifunctional ion channel with intrinsic kinase activity and is ubiquitously expressed in the animal/human body. Accumulated knowledge of TRPM7 suggests that it plays an essential role in normal physiological processes, including the development, survival, proliferation, differentiation, and migration of cells. The aim of this study was to demonstrate the presence and expression patterns of TRPM7 in normal canine mammary glands using reverse transcription-polymerase chain reaction (RT-PCR), Western blotting, and immunohistochemistry. Normal mammary gland tissue samples were obtained from five female beagle dogs. RT-PCR and sequencing of the amplified PCR products demonstrated the presence of TRPM7 mRNA in normal mammary glands, and the presence of TRPM7 protein was confirmed by Western blotting. Immunohistochemical investigations demonstrated the expression of TRPM7 in the apical membrane of acinar and ductal epithelial cells in the canine mammary glands. These results provide the first evidence of the presence and distribution of TRPM7 in the canine mammary gland and could help explain the physiological and pathological roles of TRPM7 in the canine mammary gland; however, additional studies are required to elucidate these roles.

NEP1-40-overexpressing neural stem cells enhance axon regeneration by inhibiting Nogo-A/NgR1 signaling pathway

2021 ◽  
Vol 18 ◽  
Author(s):  
Bi Zhang ◽  
Dalin Wang ◽  
Xusheng Li ◽  
Shengsen Yang ◽  
Haifeng Yuan
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Signaling Pathway ◽  
Western Blotting ◽  
Axon Regeneration ◽  
Rt Pcr ◽  
Pregnant Rat ◽  
Protein Levels ◽  
Cord Injury ◽  
Antagonistic Peptide

Background: Nogo-66 antagonistic peptide (NEP1-40) offers the potential to improve spinal cord injury (SCI). Objective: To explore the effect of NEP1-40 overexpression on neural stem cells (NSCs) regulating the axon regeneration of injured neurons. Methods: We isolated NSCs from brain tissues of pregnant rat fetuses and used Nestin immunofluorescence to identify them. The NEP1-40 overexpressing NSCs were constructed by transfection with the NEP1-40-overexpressing vector. The expression of NSCs differentiation associated markers including Tuj-1, GFAP, Oligo2 and MBP, were detected by RT-PCR, western blotting and immunofluorescence. NeuN immunofluorescence staining was used to measure the number of neurons. And western blotting was used to detect the phosphorylation levels of LIMK1/2, cofilin and MLC-2 and the protein levels of GAP-43, MAP-2 and APP. Results: The NEP1-40 overexpression promoted the expression level of Tuj-1, Oligo2 and MBP, and increased the number of Tuj-1, Oligo2 and MBP positive cells. NEP1-40-overexpressing NSCs (NEP-NSCs) improved NeuN positive cells of co-culture with injured neurons. And NEP-NSCs also increased the protein levels of axon regeneration indicators (GAP-43, MAP-2) and decreased APP protein level. In addition, the phosphorylation level of LIMK1/2, cofilin and MLC-2 were markedly decreased in NEP-NSCs. Conclusion: NEP1-40 overexpression enhanced the ability of NSCs differentiation into neurons and promoted axon regeneration by inhibiting the Nogo-A/NgR1 signaling pathway. This study provides an alternative gene modified transplantation NSCs for the SCI treatment.

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