This assay was designed to construct the prokaryotic expression vector, investigate the expression of PEBP-like in Escherichia coli and purify its product. The PEBP gene was inserted into the vector pET30a (+). The recombinant vector was transferred into E. coli BL21 (DE3)and induced the expression of protein by low concentration of IPTG and low temperature overnight. After purification, the supernatants were analyzed by SDS-PAGE and the results were identified by Western blotting. After IPTG induction, a new anticipating fusion protein of 28 kD appeared as an expected size, and its product was 26.8% in total protein, the fusion protein was positive by Western blotting. The prokaryotic expression system of PEBP-like is successfully constructed. It lays the foundation for the further application study on the antifreeze characters of the PEBP.
Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Gene Expression Regulation, Plant ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Plant Proteins ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Saussurea ; genetics

OBJECTIVE: To detect the expression of Raf kinase inhibitor protein (RKIP) and epithelial cadherin (E-cadherin) in human prostate cancer tissues, and their correlation. METHODS: We discussed the relationship between RKIP and E-cadherin and the clinical stage and pathological classification of prostate cancer by immunofluorescence histochemistry staining in the test of expression of RKIP in 26 prostate cancer tissues and 14 BPH tissues, and analyzed the correlation between them. RESULTS: The expression of RKIP and E-cadherin in prostate cancer tissues was obviously lower than that in the benign prostatic hypertrophy tissues. The expression of RKIP and E-cadherin in the dys-good differentiation group (Gleason 8-10) was significantly lower than that in the good differentiation group(GleasonCONCLUSION RKIP and E-cadherin are metastasis suppressor factors, whose decreased expression can increase the invasive capability of prostate cancer and suppress its differentiation. RKIP can suppress the metastasis of prostate cancer by increasing the E-cadherin expression in prostate cancer.
Adenocarcinoma ; metabolism ; pathology ; Aged ; Aged, 80 and over ; Cadherins ; genetics ; metabolism ; Fluorescent Antibody Technique ; Humans ; Male ; Middle Aged ; Neoplasm Metastasis ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Prostatic Neoplasms ; metabolism ; pathology

The Raf kinase inhibitory protein (RKIP) has been demonstrated to modulate different intracellular signaling pathways in cancers. Studies have shown that RKIP is frequently downregulated in cancers; therefore, attempts have been made to upregulate the expression of RKIP using natural and synthetic agents for the treatment of human malignancies. Moreover, various regulators such as specific proteins and microRNAs (miRNAs) that are involved in the regulation of RKIP expression have also been identified. RKIP mechanistically modulates the apoptotic regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) signaling. Because of its critical role in human cancers, RKIP has drawn much research attention, and our understanding is expanding rapidly. Here, we summarize some of the biological complexities of RKIP regulation. However, we restrict our discussion to selected tumors by focusing on TRAIL, miRNAs and natural agents. Emerging evidence suggests a role for natural agents in RKIP regulation in cancer cells; therefore, naturally occurring agents may serve as cancer-targeting agents for cancer treatment. Although the literature suggests some advancement in our knowledge of RKIP biology, it is incomplete with regard to its preclinical and clinical efficacy; thus, further research is warranted. Furthermore, the mechanism by which chemotherapeutic drugs and novel compounds modulate RKIP and how nanotechnologically delivered RKIP can be therapeutically exploited remain to be determined.
Apoptosis ; Gene Expression Regulation, Neoplastic ; Humans ; Male ; MicroRNAs/genetics ; Neoplasms/genetics/*metabolism ; Phosphatidylethanolamine Binding Protein/genetics/*metabolism ; Protein Interaction Maps ; *Signal Transduction ; TNF-Related Apoptosis-Inducing Ligand/genetics/metabolism

OBJECTIVETo study the development of changes for Raf kinase inhibitor protein (RKIP) and its mRNA in rats hippocampus after electromagnetic radiation.

METHODSRats were exposed to X-band high power microwave (X-HPM), S-band high power microwave (S-HPM) and electromagnetic pulse (EMP) radiation source respectively. The animal model of electromagnetic radiation was established. Western blot was used to detect the expression of RKIP, and RT-PCR was applied to detect the expression of RKIP mRNA. The interaction of RKIP and Raf-1 was measured with co-immunoprecipitation method, and the expression of cerebral choline acetyltransferase (CHAT) was measured by immunohistochemistry.

RESULTSThe expression of RKIP significantly down-regulated at 6 h after radiation, and recovered at 1 d in group EMP, but the down-regulation continued during 1 approximately 7 d after radiation in the two microwave groups. The expression of RKIP mRNA changed wavily during 6 h approximately 7 d after radiation, which showed down-regulation at 6 h, and up-regulation at 3 d. The interaction of RKIP and Raf-1 decreased during 6 h approximately 7 d after radiation, most significantly at 7 d, and the two microwave groups were more significant. The expression of CHAT decreased continuously during 6 h approximately 7 d after radiation, and generally recovered on 14 d.

CONCLUSIONThe down-regulation of RKIP and its related proteins of hippocampus is induced by electromagnetic radiation.

Animals ; Electromagnetic Radiation ; Hippocampus ; metabolism ; radiation effects ; MAP Kinase Kinase Kinases ; metabolism ; Male ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Proto-Oncogene Proteins c-raf ; RNA, Messenger ; genetics ; Rats ; Rats, Wistar

OBJECTIVETo investigate the expression of RKIP, p65 and pERK in hepatocellular carcinoma (HCC) and theIr correlation with invasion and metastasis of HCC.

METHODSReverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of RKIP mRNA. The expression levels of RKIP, p65 and pERK proteins in HCC tumor and peritumoral tissues were determined by immunohistochemistry and Western blot analysis. Statistical analysis was performed to determine the relationship between their expression and clinicopathological parameters.

RESULTSRKIP protein expression level (RKIP/actin) was 0.579 ± 0.380 in HCCs, 1.178 ± 0.659 in peritumoral tissues and 1.115 ± 0.442 in normal liver tissues. The pERK protein level was 1.023 ± 0.478, 0.605 ± 0.367 and 0.461 ± 0.293, p65 protein level was 0.83 ± 0.376, 0.63 ± 0.337 and 0.466 ± 0.345, respectively. Immunohistochemistry analysis showed that the RKIP positive rates in HCCs, peritumoral tissues and normal liver tissues, were 22.2%, 86.0%, and 93.8%, positive rates of p65 were 73.6%, 56.0% and 37.5%, positive rates of pERK were 65.3%, 38.0% and 31.3%, respectively. Statistical analysis revealed that there was a significant difference in RKIP protein expression levels (P < 0.05), but no significant difference in RKIP mRNA expression levels (P > 0.05) among HCC tumors, peritumoral tissues and normal liver tissues. The p65-positive and pERK-positive rates were higher in tumor tissues than that in peritumoral tissues and in normal liver tissues (P < 0.05), but RKIP-positive rates were lower in tumor tissues than that in paritumoral tissues and normal liver tissues (P < 0.05). RKIP protein expression levels were significantly lower in HCCs with intrahepatic or lymphatic metastasis than that in without. The RKIP positive rates in moderately and well differentiated HCCs were significantly higher than that in poorly differentiated HCCs. There was a relationship between RKIP and pERK expressions (P = 0.04), but RKIP expression was not correlated with p65 expression in HCCs (P = 0.143).

CONCLUSIONSOur findings indicate that the down-regulation of RKIP expression may serve as a predictive marker for HCC development, progression and metastasis, which may contribute to the elevated ERK activity. The inhibiting effect of RKIP on invasion and metastasis of liver cancer cells may be due to the down-regulation of pERK expression rather than p65 expression.

Adult ; Aged ; Carcinoma, Hepatocellular ; metabolism ; pathology ; Down-Regulation ; Extracellular Signal-Regulated MAP Kinases ; metabolism ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Liver ; metabolism ; Liver Neoplasms ; metabolism ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Invasiveness ; Phosphatidylethanolamine Binding Protein ; genetics ; metabolism ; Phosphorylation ; RNA, Messenger ; metabolism ; Transcription Factor RelA ; metabolism