Determination of the Relative Expression Levels of Rubisco Small Subunit Genes in Arabidopsis by Rapid Amplification of cDNA Ends

2001 ◽  
Vol 291 (2) ◽  
pp. 237-244 ◽  
Author(s):  
Minsoo Yoon ◽  
Joanna J. Putterill ◽  
Gavin S. Ross ◽  
William A. Laing
Keyword(s):  
Small Subunit ◽  
Expression Levels ◽  
Relative Expression ◽  
Rubisco Small Subunit ◽  
Rapid Amplification

scholarly journals Establishment and Fractionation of Metastatic Axillary Lymph Node Cell Suspension for Determination of Protein Expression Levels of Nuclear cFOS and Cytosolic TGFβ1 from Breast Cancer Patients

2021 ◽  
Author(s):  
Vesna Ivanović ◽  
Nasta Dedović-Tanić ◽  
Zorka Milovanović ◽  
Bratislav Stojiljković ◽  
Miroslav Demajo ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Lymph Node ◽  
Protein Expression ◽  
Cell Suspension ◽  
Axillary Lymph Node ◽  
Transforming Growth Factor ◽  
Axillary Lymph ◽  
Expression Levels ◽  
Nodal Cell

Abstract Background. Metastatic Axillary Lymph Node (mALN) status is currently the most important prognostic factor in the management of primary breast cancer (BC). Thus, development of specimens which enable identification of new mALN markers, involved in the progression of the disease, are of considerable interest. The specific aim of this work was to describe the method of establishment of Metastatic Axillary Nodal Cell Suspension and its fractionation, termed Fractionated Nodal Cell Suspension (FNCS), into nuclear and cytosolic extracts to enable determination of protein expression levels of nuclear cFOS and cytosolic Transforming Growth Factor β1 (TGFβ1) in BC patients. Results. To standardize the procedure, HeLa cells were successfully fractionated into nuclear/cytosolic extracts with confirmed presence of nuclear cFOS and cytosolic TGFβ1 proteins. Subsequently, the ALN Cell Suspension specimens were obtained and further fractionated from a pilot sample of six ALN tissue pairs, mALN versus autologous normal ALN (nALN), dissected from invasive BC patients. The mALN results revealed statistically significant overexpression in nuclear cFOS and cytosolic TGFβ1 protein levels, which was proportional to the respective values of mALN diameter of tumor deposits. Conclusions. Detailed protocol for establishment and fractionation of mALN cell suspension specimens, termed FNCS, into nuclear and cytosolic extracts is here described for the first time. This approach might be a convenient ex vivo model for simultaneous analysis of protein, RNA and DNA biomarkers from nuclear/cytosolic extracts of the same mALN tissue sample. It might have potential to enable, in the age of genomics and personalized medicine, an identification of novel mALN biomarkers and thus improve the screening, diagnosis and prognosis of invasive BC.

scholarly journals Relationship between miR-338-3p and Clinicopathological Parameters, Prognosis, and STAT3 mRNA Expression in Nasopharyngeal Carcinoma

2021 ◽  
Vol 2021 ◽  
pp. 1-7
Author(s):  
Youyou Wang ◽  
Huijun Ren ◽  
Zhaohu Pan ◽  
Ben Liu ◽  
Fan Lin
Keyword(s):  
Survival Rate ◽  
Nasopharyngeal Carcinoma ◽  
Mrna Expression ◽  
Distant Metastasis ◽  
Tnm Stage ◽  
Control Group ◽  
Expression Levels ◽  
Relative Expression ◽  
T Stage ◽  
The Relationship

Objective. To investigate the expression of miR-338-3p in nasopharyngeal carcinoma (NPC) and its relationship with STAT3 mRNA expression as well as their relationship with clinical pathological parameters and prognosis of patients. Methods. From September 2016 to September 2018, 71 patients with NPC were selected as the NPC group, and 71 samples of NPC tissues were collected during the operation. A total of 23 patients who underwent biopsy due to chronic nasopharyngitis were selected as the control group and 23 nasopharyngeal mucosal tissues were collected. The expressions of miR-338-3p and STAT3 mRNA in nasopharyngeal tissue of two groups were detected by real-time quantitative PCR, and the relationship between the two was analyzed. To collect clinical data of NPC patients and analyze the relationship between the expressions of miR-338-3p and STAT3 in NPC tissues and clinical pathological parameters of the patients, we followed up the patients with nasopharyngeal carcinoma for three years to observe the relationship between miR-338-3p, STAT3, and the prognosis of the patients. Results. The relative expression levels of miR-338-3p in nasopharyngeal tissues of the NPC group and the control group were 0.39 ± 0.05 and 1.01 ± 0.09, respectively ( P  < 0.05). The relative expression levels of STAT3 mRNA in nasopharyngeal tissues of the NPC group and the control group were 3.82 ± 0.21 and 1.04 ± 0.11, respectively ( P  > 0.05). miR-338-3p was negatively correlated with the relative expression of STAT3 mRNA in nasopharyngeal carcinoma (r = 0.038, P  > 0.05). The expression of miR-338-3p was related to the age of the patient, clinical TNM stage, T stage, and distant metastasis (all P  < 0.05). STAT3 expression was correlated with clinical TNM stage, T stage, and distant metastasis in our patient ( P  < 0.05). The expressions of miR-338-3p and STAT3 in nasopharyngeal carcinoma tissues from different gender, histological type, N stage, M stage, and degree of differentiation showed no statistical differences ( P  > 0.05). The survival rate of the group with low miR-338-3p expression was significantly lower than that of the group with high miR-338-3p expression ( P  > 0.05). The survival rate of patients with the high STAT3 expression group was significantly lower than that of patients with the low STAT3 expression group ( P  > 0.05). Conclusion. There is a negative correlation between the low expression of miR-338-3p and the high expression of STAT3 in NPC, which are all related to the TNM stage, T stage, and prognosis of the patient.

scholarly journals Folate Reverses NF-κB p65/Rela/IL-6 Level Induced by Hyperhomocysteinemia in Spontaneously Hypertensive Rats

2021 ◽  
Vol 12 ◽  
Author(s):  
Lihua Zhang ◽  
Zhongliang Li ◽  
Changcheng Xing ◽  
Ning Gao ◽  
Rui Xu
Keyword(s):  
Oxidative Stress ◽  
Hypertensive Rats ◽  
Spontaneously Hypertensive ◽  
Expression Levels ◽  
Relative Expression ◽  
Abnormal Metabolism ◽  
Rat Thoracic Aorta ◽  
Immune Inflammation ◽  
Arterial Damage ◽  
Rat Tail

Hyperhomocysteinemia (HHcy) is derived from the abnormal metabolism of homocysteine (Hcy) and is related to metabolic-related diseases. In addition, HHcy combined with hypertension increases the risk of cardiovascular diseases (CVD). However, the mechanism of HHcy aggravating hypertensive arterial damage and the efficacy of folate (FA) as a beneficial supplement have not been fully elucidated. In this study, we established a rat HHcy model and a hypertension combined with HHcy model. Rat tail artery blood pressure (BP), plasma Hcy, serum superoxide dismutase (SOD), and malondialdehyde (MDA) were measured. Rat thoracic aorta was for pathological analysis after 12 weeks of the experiment. The relative expression levels of oxidative stress and immune/inflammation in rat arterial tissues were detected by quantitative real-time polymerase chain reaction (qRT-PCR) and western blotting. The results demonstrated that the relative expression levels of oxidative stress and immune/inflammation were the highest in the hypertension combined with HHcy group, followed by the hypertension group. Compared with the hypertension group, the hypertension combined with HHcy group up-regulated the expression levels of interleukin-6 (IL-6) and nuclear factor-κ-gene binding (NF-κB) p65/Rela, but not NADPH oxidase (Nox). Furthermore, folate inhibited the expression of IL-6 and NF-κB p65/Rela, reduced the levels of MDA and HHcy, but significantly increased the SOD level. In conclusion, HHcy synergistically aggravated the arterial damage factor of hypertension through immune/inflammatory response. However, folate demonstrated anti-inflammatory properties and reversed the NF-κB p65/Rela/IL-6 level induced by HHcy in hypertensive rats.

scholarly journals iTRAQ-Based Sperm Proteomic Analyses Reveals Candidate Proteins Affecting the Quality of Spermatozoa From Boar on Plateaus

2020 ◽  
Author(s):  
Yanling Zhao ◽  
Yaomei Wang ◽  
Feipeng Guo ◽  
Bo Lu ◽  
Jiale Sun ◽  
...  
Keyword(s):  
Western Blot ◽  
Cellular Response ◽  
Sperm Quality ◽  
Semen Analysis ◽  
Estrogen Signaling ◽  
Glutathione Metabolism ◽  
Computer Assisted ◽  
Expression Levels ◽  
Relative Expression

Abstract Background: Tibetan pigs (TP) exhibit heritable adaptations to their hypoxic environments as a result of natural selection. Whereas, what candidate proteins affecting the sperm quality of boar on plateaus has not been clearly investigated yet. Methods: In this study, to reveal the candidate proteins affecting the quality of spermatozoa from boar on plateaus, we analyzed the sperm quality by Computer-assisted semen analysis (CASA) system and Reactive oxygen species (ROS) levels, compared the sperm proteomes between TP and Yorkshire pigs (YP) raised at high altitudes using the isobaric tags for relative and absolute quantitation (iTRAQ) in combination with the liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomic method, and confirmed the relative expression levels of the four proteins by western blot. Results: The sperm quality of the TP was superior to that of the YP on plateaus. Of 1,555 quantified proteins, 318 differentially expressed proteins (DEPs) were identified. Gene ontology (GO) analysis revealed that the DEPs were predominantly associated with the sorbitol metabolic process, removal of superoxide radicals, cellular response to superoxide, response to superoxide and regulation of the mitotic spindle assembly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were mainly enriched in pathways involved in the regulation of the actin cytoskeleton, glutathione metabolism, oxidative phosphorylation, and estrogen signaling. And based on the protein-protein interaction (PPI) network analysis, we identified 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) that might play important roles that affect the sperm quality of boar on plateaus. Moreover, the relative expression levels of the proteins (CFL1, EGF, FN1, and GPX4) were confirmed by western blot. Conclusions: Our results reveal 8 candidate proteins (FN1, EGF, HSP90B1, CFL1, GPX4, NDUFA6, VDAC2, and CP) affecting the sperm quality of boar on plateaus, providing a reference for studies on improving sperm quality and the molecular breeding of boar on plateaus.

scholarly journals Expression and clinical significance of PTPN12 in clear cell renal cell carcinoma

2020 ◽  
Vol 48 (12) ◽  
pp. 030006052093604
Author(s):  
Yi Jin ◽  
Tian-xi Wang ◽  
Hao Li ◽  
Peng Guo ◽  
Qing-qing Wang
Keyword(s):  
Renal Cell Carcinoma ◽  
Cell Carcinoma ◽  
Mrna Expression ◽  
Renal Cell ◽  
Clear Cell ◽  
Cell Renal Cell Carcinoma ◽  
Expression Levels ◽  
Relative Expression ◽  
Normal Tissues ◽  
Mrna Expression Levels

Background Clear cell renal cell carcinoma (ccRCC) is a common urological disease. Expression of the protein tyrosine phosphatase 12 gene ( PTPN12) is decreased in many cancers; however, the relationship between PTPN12 gene function and renal cancer remains unclear. Methods We detected PTPN12 protein expression in ccRCC and corresponding normal tissues from 64 patients with ccRCC by immunohistochemistry, and relative PTPN12 mRNA levels by real-time quantitative polymerase chain reaction. The relationships between the relative expression levels of PTPN12 mRNA and the patients’ clinical data were analyzed. Results PTPN12 protein and mRNA expression levels were significantly lower in ccRCC compared with the corresponding normal tissues. The mRNA expression levels in the ccRCC and corresponding normal tissues from the 64 patients with ccRCC were 0.459±0.445 and 1.001±0.128, respectively, compared with the control (glyceraldehyde 3-phosphate dehydrogenase). There was a significant correlation between relative expression of PTPN12 mRNA in ccRCC tissues and tumor diameter and clinical stage. Conclusion The expression levels of PTPN12 protein and mRNA were significantly lower in ccRCC tissues compared with normal tissues. The role of PTPN12 may provide new insights and evidence to aid the diagnosis and targeted therapy of ccRCC.

scholarly journals Circ_MDM2_000139, Circ_ATF2_001418, Circ_CDC25C_002079, and Circ_BIRC6_001271 Are Involved in the Functions of XAV939 in Non-Small Cell Lung Cancer

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Haixiang Yu ◽  
Lei Xu ◽  
Zhengjia Liu ◽  
Bo Guo ◽  
Zhifeng Han ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
Cell Lung Cancer ◽  
Regulatory Network ◽  
Family Members ◽  
Enrichment Analysis ◽  
Small Cell ◽  
Small Cell Lung ◽  
Expression Levels ◽  
Relative Expression

Background. The small molecule inhibitor XAV939 could inhibit the proliferation and promote the apoptosis of non-small cell lung cancer (NSCLC) cells. This study was conducted to identify the key circular RNAs (circRNAs) and microRNAs (miRNAs) in XAV939-treated NSCLC cells. Methods. After grouping, the NCL-H1299 cells in the treatment group were treated by 10 μM XAV939 for 12 h. RNA-sequencing was performed, and then the differentially expressed circRNAs (DE-circRNAs) were analyzed by the edgeR package. Using the clusterprofiler package, enrichment analysis for the hosting genes of the DE-circRNAs was performed. Using Cytoscape software, the miRNA-circRNA regulatory network was built for the disease-associated miRNAs and the DE-circRNAs. The DE-circRNAs that could translate into proteins were predicted using circBank database and IRESfinder tool. Finally, the transcription factor (TF)-circRNA regulatory network was built by Cytoscape software. In addition, A549 and HCC-827 cell treatment with XAV939 were used to verify the relative expression levels of key DE-circRNAs. Results. There were 106 DE-circRNAs (including 61 upregulated circRNAs and 45 downregulated circRNAs) between treatment and control groups. Enrichment analysis for the hosting genes of the DE-circRNAs showed that ATF2 was enriched in the TNF signaling pathway. Disease association analysis indicated that 8 circRNAs (including circ_MDM2_000139, circ_ATF2_001418, circ_CDC25C_002079, and circ_BIRC6_001271) were correlated with NSCLC. In the miRNA-circRNA regulatory network, let-7 family members⟶circ_MDM2_000139, miR-16-5p/miR-134-5p⟶circ_ATF2_001418, miR-133b⟶circ_BIRC6_001271, and miR-221-3p/miR-222-3p⟶circ_CDC25C_002079 regulatory pairs were involved. A total of 47 DE-circRNAs could translate into proteins. Additionally, circ_MDM2_000139 was targeted by the TF POLR2A. The verification test showed that the relative expression levels of circ_MDM2_000139, circ_CDC25C_002079, circ_ATF2_001418, and circ_DICER1_000834 in A549 and HCC-827 cell treatment with XAV939 were downregulated comparing with the control. Conclusions. Let-7 family members and POLR2A targeting circ_MDM2_000139, miR-16-5p/miR-134-5p targeting circ_ATF2_001418, miR-133b targeting circ_BIRC6_001271, and miR-221-3p/miR-222-3p targeting circ_CDC25C_002079 might be related to the mechanism in the treatment of NSCLC by XAV939.

scholarly journals Effects of Disruption of Five FUM Genes on Fumonisin Biosynthesis and Pathogenicity in Fusarium proliferatum

Toxins ◽  
2019 ◽  
Vol 11 (6) ◽  
pp. 327 ◽  
Author(s):  
Lei Sun ◽  
Xu Chen ◽  
Jian Gao ◽  
Yuan Zhao ◽  
Lianmeng Liu ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Fusarium Proliferatum ◽  
Expression Levels ◽  
Relative Expression ◽  
Severe Reduction ◽  
Rot Disease

The mycotoxin fumonisin is known to be harmful to humans and animals, and thus it is desirable to reduce fumonisin content in crop products. We explored the functions of several genes that function in fumonisin biosynthesis (FUM1, FUM6, FUM8, FUM19, and FUM21) in Fusarium proliferatum and found that deletion of FUM1, FUM6, FUM8, or FUM21 results in a severe reduction in fumonisin biosynthesis, while loss of FUM19 does not. In addition, fumonisin-deficient strains display significantly decreased pathogenicity. Co-cultivation of the ΔFUM1, ΔFUM6, ΔFUM8, and ΔFUM19 mutants restores fumonisin synthesis. However, co-cultivation was unable to restore fumonisin synthesis in the ΔFUM21 strain. The relative expression levels of three key FUM genes (FUM1, FUM6, and FUM8) differed significantly in each mutant strain; notably, the expression levels of these three genes were significantly down-regulated in the ΔFUM21 strain. Taken together, our results demonstrate that FUM1, FUM6, FUM8, and FUM21 are essential for fumonisin synthesis, and FUM19 is non-essential. Partial mutants lost the ability to synthesize fumonisin, the co-culture of the mutants was able to restore fumonisin biosynthesis. While the pathogenicity of F. proliferatum is affected by many factors, inhibition of the synthesis of the mycotoxin fumonisin will weaken the pathogenicity of rice spikelet rot disease (RSRD).

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