Angiogenin, as a member of the ribonuclease superfamily, is an angiogenic protein. The angiogenic ability of angiogenin plays an important role in many physical and pathological processes. Angiogenin can induce endothelial cell migration, proliferation, tubulation, as well as inhibition of cellular apoptosis. Angiogenin can be used to modulate the angiogenetic process of tissue engineered constructions via local delivery. Furthermore, angiogenin can also be regarded as a biomarker for diagnostic evaluation of malignancy, or as a target for cancer therapy. This paper presents a comprehensive overview of the angiogenic mechanisms of angiogenin, as well as its potential application in the process of wound healing and treatment of ischemic diseases and malignancy.
Cell Movement ; Humans ; Neovascularization, Pathologic ; Neovascularization, Physiologic ; Ribonuclease, Pancreatic ; therapeutic use

PURPOSE: We determined the clinical significance of telomerase activity and telomere length in breast cancer patients and also developed the measuring system of telomerase activity change with RNAse A pre-treatment. MATERIALS AND METHODS: We measured the telomerase activity in 71 breast cancer tissues and paired normal tissues with TRAP (Telomeric Repeat Amplification Protocol) assay. Telomerase activity was calculated by computer-assisted densitometry compared to telomerase activity of the 293 control cell line. To develop the measuring system of telomerase activity modulation, we measured the telomerase activity after the treatment with RNAse A, 150microgram/ml, which inhibited 70% of telomerase activity compared to control in the 293 control cell line. In 59 paired tissues with telomerase activity, terminal restriction fragment (TRFs) length were measured using Southern blotting. RESULTS: Sixty-three out of 71 cancer tissues showed telomerase activity (88.7%), while no telomerase activity was detected in their paired normal tissues. Telomerase activity was correlated to the node metastasis (p=0.02) and stage (p=0.005), but not to the tumor size or the hormonal receptor status. TRFs were neither specific to tumor tissues nor related to any of the clinical parameters. However, changes of TRFs of the tumor tissues from their paired normal tissues were correlated to the telomerase activities. Also the patients with different TRFs between cancer and normal tissues were in more advanced stage. After pre-treatment with the 150microgram/ml of RNAse A, telomerase activity in the tumor tissues showed variable inhibition. Relative inhibition, the ratio of inhibited telomerase activity in each tumor tissue compared to the inhibition of 293 control cell line, was proportional to the telomerase activity. CONCLUSION: In breast cancer, telomerase activity was specific to the tumor tissues and correlated to tumor progression. A combination of telomerase activity and TRFs changes can be used as a guidline in detecting a better candidate for telomerase inhibition. Semi-quantitative assay with RI system can be used in evaluating the changes of telomerase activity after treatment with a new telomerase inhibitor with TRAP assay.
Biological Therapy* ; Blotting, Southern ; Breast Neoplasms* ; Breast* ; Cell Line ; Densitometry ; Humans ; Neoplasm Metastasis ; Ribonuclease, Pancreatic ; Telomerase* ; Telomere

OBJECTIVEEstablish the fluorescent quantitative RT-PCR detection method for rabies virus (RV) and construct RNase-resistant virus-like particles as positive controls.

METHODSAnalyze the database in GenBank, the probe and the primers were designed in the conservative region of N gene of rabies virus and the method of real-time fluorescent quantitative PCR was obtained; On the basis of MS2 phage, with the technology of gene recombination, prepare the RNase-resistant virus-like particles for RV positive controls;

RESULTSRNase-resistant virus-like particles were obtained after prokaryotic expression in E. coli. The designed primers and probe were confirmed to be very specific and conservative, and be sensitive to-concentration of 15 copies/microl.

CONCLUSIONEstablished the method of detecting rabies virus by reverse transcription real-time quantitative PCR,obtained the RNase-resistant and no infectivity virus-like particles as positive controls of rabies virus.

Animals ; DNA Probes ; DNA, Viral ; Dogs ; Humans ; RNA, Viral ; analysis ; Rabies ; virology ; Rabies virus ; chemistry ; Reverse Transcriptase Polymerase Chain Reaction ; methods ; Ribonuclease, Pancreatic ; metabolism ; Viral Load

OBJECTIVETo express, purify and refold recombinant luteinizing hormone releasing hormone-angiogenin (LHRH-Ang) toxin using E. coli. expression system.

METHODSRecombinant LHRH-Ang expression vector was constructed by replacing of EGF fragment in plasmid pET28a/EGF-Ang with LHRH-PII fragment amplified from plasmid pET28/MSH-PE40. DNA sequencing would be used to verify the correction of fused LHRH-PII-Ang gene. Then, E. coli strain BL21 (DE3) was transformed by pET28a/LHRH-Ang vector. Expression of recombinant LHRH-Ang toxin was induced by Isopropyl-β-D-Thiogalactoside (IPTG). Refolding effects of gradient dialysis was evaluated by SDS-PAGE.

RESULTSProkaryotic expression vector pET28a/LHRH-Ang, containing LHRH-PII-Ang fusion gene, was constructed by PCR amplification, restriction enzyme digestion and ligation method. Sequence correction of fusion gene was confirmed by DNA sequencing. After IPGT induction, recombinant LHRH-Ang protein was expressed in BL21 (DE3) as inclusion body, it took 18.43% of total protein. Inclusion body was resolved in 8 mol/L urea and purified by DEAE-Sepharose FF column, the purity was 85%. Recombinant LHRH-Ang toxin was refolded and concentrated by gradient dialysis and PEG 20000, respectively.

CONCLUSIONSRecombinant LHRH-Ang protein was expressed in E. coli and refolded successfully.

Escherichia coli ; metabolism ; Gene Expression ; Genetic Vectors ; Gonadotropin-Releasing Hormone ; biosynthesis ; genetics ; Plasmids ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Ribonuclease, Pancreatic ; biosynthesis ; genetics

OBJECTIVETo explore the mechanism of electroacupuncture (EA) in improving ischemic stroke.

METHODSA Wistar rat model of focal cerebral ischemia reperfusion was made by filament occlusion. The rats were randomly divided into a normal group, a model group, an EA group. EA was given at bilateral "Hegu" (LI 4) in the EA group. Vascular endothelial growth factor (VEGF) mRNA was detected with in situ hybridization and expression of angiogenin-1 (Ang-1) and endostatin proteins with immunohistochemical method.

RESULTSThe expressions of angiogenic growth factors including VEGF and Ang-1 in the EA group were significantly increased, while the expressions of endostatin was significantly decreased as compared with those in the model group (both P<0.05).

CONCLUSIONEA improving ischemic stroke is carried out possibly through up-regulating the expression of angiogenic growth factors and down-regulating the expression of antiangiogenic growth factors.

Animals ; Brain ; metabolism ; Brain Ischemia ; metabolism ; therapy ; Electroacupuncture ; Endostatins ; analysis ; Immunohistochemistry ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Reperfusion ; Ribonuclease, Pancreatic ; analysis ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVE: Activities of nucleases (acid DNase and neutral RNase) and RNase inhibitor known to be involved in carcinogenesis and suppression of cancer were determined in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma and were compared with those of the controls. Also studied were nucleases and RNase inhibitor isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients to evaluate the properties and interactions between them. METHOD: Activities of nucleases and RNase inhibitor were measured in cancer tissue, serum and ascitic fluid of patients with hepatocellular carcinoma by ultraviolet spectrophotometry. Nucleases and RNase inhibitor were isolated from hepatocellular carcinoma tissue and ascitic fluid of the cancer patients by DEAE-cellulose column chromatography. As controls, normal tissue of the cancer patients, serum of healthy persons and ascitic fluid of cirrhotic patients were used. RESULT: Activities of DNase, RNase and RNase inhibitor were significantly increased in hepatocellular carcinoma tissue. DNase activity was not detected, RNase activity was increased and RNase inhibitor activity was unchanged in both serum and ascitic fluid of the hepatocellular carcinoma patients. DNase was isolated as a single enzyme and RNase as seven isozymes from the hepatocellular carcinoma tissue. The DNase isolated preferentially cleaved ds DNA over ss DNA and was endonuclease in nature (majority of hydrolytic products of DNA by the DNase were oligodeoxyribonucleotides). Of seven RNase isozymes isolated from the hepatocellular carcinoma tissue, isozyme I exhibited nonsecretory nature of RNase and other six isozymes secretory nature of the enzyme. Activity of RNase isozyme V was greatly increased and the activity of inhibitor complexed with the isozyme V was also increased. RNase in ascitic fluid of the cancer patient was separated into four isozymes, of which isozyme I exhibited mixed form of secretory and nonseretory nature and greatly increased in its activity. RNase isozyme V isolated in the hepatocellular carcinoma tissue was not detected in the ascitic fluid. CONCLUSION: The use of the nucleases and the inhibitor in the cancer tissue as biochemical markers for the hepatocellular carcinoma was suggested. RNase was released into the body fluid from the cancer tissue and could be used as a diagnostic marker for the hepatocellular carcinoma. An important role of the DNase in carcinogenesis of the liver was suggested. RNase isozyme V was limited in the cancer tissue and RNase isozyme I and V and inhibitors associated with these isozymes might be involved in carcinogenesis processes, suppression of cancer and maintenance of hepatocellular carcinoma through their interactions.
Ascitic Fluid ; Biomarkers ; Body Fluids ; Carcinogenesis ; Carcinoma, Hepatocellular* ; Chromatography ; DEAE-Cellulose ; Deoxyribonucleases* ; DNA ; Humans ; Isoenzymes ; Liver ; Ribonuclease, Pancreatic ; Ribonucleases* ; Spectrophotometry, Ultraviolet

PURPOSE: To investigate the properties of angiogenin (ANG) as a potential tool for the diagnosis and grading of dry eye syndrome (DES) by analyzing tear protein profiles. METHODS: Tear samples were collected with capillary tubes from 52 DES patients and 29 normal individuals as controls. Tear protein profiles were analyzed with an immunodot blot assay as a screening test. To confirm that the tear ANG levels were in inverse proportion to the disease severity grade, the ANG and lactoferrin (LF) tear contents of normal controls and DES patients were compared in an enzyme-linked immunosorbent assay. RESULTS: In the immunodot blot assay, the ANG area was lower in patients with grades 3 and 4 DES than in normal controls. The areas of basic fibroblast growth factor, transforming growth factor β2, and interleukin 10 were significantly greater than those of normal controls only in grade 4 DES patients, but these proteins were not linearly correlated with dry eye severity. Upon enzyme-linked immunosorbent assay analysis, the mean concentrations of ANG and LF decreased significantly as dry eye severity increased, except between grades 1 and 2. In addition, the ratios of ANG and LF to total tear proteins were correlated significantly with DES severity. CONCLUSIONS: ANG level was significantly lower in DES patients than in normal controls, and was significantly correlated with the worsening severity of DES, except between grades 1 and 2, as was LF. Therefore, ANG may be a useful measure of DES severity through proteomic analysis.
Adult ; Aged ; Angiogenesis Inducing Agents/pharmacology ; Dry Eye Syndromes/*diagnosis/metabolism ; Enzyme-Linked Immunosorbent Assay ; Female ; Follow-Up Studies ; Humans ; Immunoblotting ; Male ; Middle Aged ; Proteomics/methods ; Ribonuclease, Pancreatic/*pharmacology ; Severity of Illness Index ; Tears/chemistry ; Young Adult

Angiogenin, a potent inducer of angiogenesis, is expressed in human endometrium. This study was performed to compare the expression of angiogenin mRNA level in the eutopic endometrium from women with and without endometriosis. Thirty-two women with advanced stage endometriosis and 29 control women were recruited. Following isolation of total RNA from endometrial tissue and reverse transcription, cDNA samples were amplified by real time polymerase chain reaction to quantify the expression of angiogenin genes. In selected patients, immunohistochemical staining was utilized to localize the area of angiogenin expression. Angiogenin mRNA level was significantly lower in the endometriosis group than in the control group during the secretory phase, especially the mid-secretory phase, and the decline was observed mainly in the women who presented with infertility. Within the endometriosis group, angiogenin mRNA levels did not differ between the proliferative and secretory phases, but, in the control group, the level in the secretory phase was higher than that during the proliferative phase. Immunohistochemistry showed that the glandular epithelial cell layer was decorated positively in both groups. These findings suggest that the relative deficiency of angiogenin expression in the secretory endometrium could impair implantation in women with advanced stage endometriosis.
Adult ; Endometriosis/*metabolism/pathology ; Endometrium/*metabolism ; Female ; Fertility ; *Gene Expression Regulation ; Humans ; Immunohistochemistry/methods ; Menstrual Cycle ; Models, Biological ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Ribonuclease, Pancreatic/*biosynthesis

The aim of study was to investigate the expression of angiogenin-2 (Ang-2) and vascular endothelial growth factor (VEGF) expression in acute leukemia (AL) and its significance in the angiogenesis and progress of AL. Serum levels of Ang-2 and VEGF in 24 de novo, 18 complete remitted (CR), 7 unremitted, 13 relapsed patients with AL were measured by enzyme-linked immunosorbent assay (ELISA), and compared with those of normal controls. The results showed that the serum levels of Ang-2 and VEGF in de novo, unremitted and relapsed patients were significantly higher than those in normal controls (p < 0.01). Compared with de novo group, the serum levels of Ang-2 and VEGF in CR patients decreased significantly, but showing no significant difference from those in normal controls (p > 0.05). In relapsed patients, the serum levels of Ang-2 and VEGF were obviously higher than those in unremitted and CR patients (p < 0.01). It is concluded that the expressions of Ang-2 and VEGF are closely related with occurrence and development of acute leukemia, the VEGF may regulate Ang-2 expression and promote angiogenesis and acute leukemia development. Preventing expressions of Ang-2 and VEGF may seem as targets for leukemia therapy in the future.
Acute Disease ; Adolescent ; Adult ; Aged ; Case-Control Studies ; Child ; Female ; Humans ; Leukemia, Myeloid, Acute ; metabolism ; pathology ; Male ; Middle Aged ; Neovascularization, Pathologic ; pathology ; Ribonuclease, Pancreatic ; metabolism ; Vascular Endothelial Growth Factor A ; metabolism ; Young Adult

This study was purposed to investigate the angiogenin (ANG) expression in COS-7 cells and its biological activity. The gene of angiogenin was obtained from mononuclear cells of peripheral blood by using RT-PCR and inserted into eukaryotic expression vector of pcDNA3.1. After being transfected into COS-7 cells, the recombinant ANG was identified by Western blot assay. The function of promoting proliferation of ANG to ECV304 cells was detected by MTT method, and its activity of vascularization was analyzed by chick embryo chorioallantois treated by the culture supernatant after transfection with pcDNA3.1-ang. The result showed that recombinant ANG was expressed in COS-7 cells after transfection for 24 to 36 hours. It could specifically react with monoclonal antibody against ANG. The recombinant ANG could obviously promote the proliferation of ECV304 cells. In contrast with the control group, the culture supernatant of pcDNA3.1-ang transfected group could stimulate the angiogenesis in embryo chorioallantois. It is concluded that the ang transiently expresses in COS-7 cells, and its expression product obviously stimulates the cell proliferation and angiogenesis.
Angiogenesis Inducing Agents ; pharmacology ; Animals ; COS Cells ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Cercopithecus aethiops ; Endothelial Cells ; cytology ; Genetic Vectors ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Ribonuclease, Pancreatic ; biosynthesis ; genetics ; pharmacology ; Transfection