Development of a novel piezoelectric-driven non-resonant elliptical vibrator with adjustable characteristics.

Aiming at the fabrication of a micro-textured surface, a novel piezoelectric-driven non-resonant elliptical vibrator is proposed in this paper; the output characteristics could be adjusted by the length change of the tool holder. The flexible mechanism is the primary structure of the vibrator, which includes a lever type mechanism, an enhanced Scott-Russell mechanism, and a T-shaped mechanism. The former two mechanisms are used to enlarge the output of the piezoelectric actuator, and the T-shaped mechanism is applied to transfer the parallel movements to the elliptical trajectory. The theoretical models including the elliptical trajectory, output stiffness, and resonant frequencies are established to investigate the impacts of the tool holder and controlling signals on the output characteristics of the vibrator, which are further validated using the finite element analysis method. A prototype is developed by integrating the non-resonant elliptical vibrator assembly and controlling system. Some experiments are carried out to verify the basic performance and the adjustable properties of the vibrator.

[Analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor activity testing].

Objective: To investigate the analytical performance verification protocols and performance specifications of platelet-dependent von Willebrand factor (VWF) activity testing (VWF:GPIbM) for clinical laboratories. Methods: According to Clinical Laboratory Standards Institute (CLSI) documents and National Health Standard of China, the performance verification of VWF:GPIbM was designed and implemented using Sysmex CS-5100 instrument and its corresponding reagents. (1) Precision verification: Two commercial quality control samples (with normal and low activity levels) and three plasma pools (with activity range from 5.0% to 150.0%) were prepared. Each sample was tested five times daily for five consecutive days. The coefficient of variation (CV) of intra-and inter-run precisions were calculated, and the precision evaluation criterion was set according to package inserts. (2) Trueness verification: The calibrator was diluted to five reference materials with activity values of 5.2%, 31.2%, 62.4%, 104.0% and 138.7%, and each reference material was tested five times daily for five consecutive days. The bias between the measured value and the reference value was calculated, and the trueness evaluation criterion was set according to the total allowable error. (3) Linearity verification: Ten pooled plasmas with theoretical value range from 3.6% to 160.4% were prepared for the linearity verification of two calibration curves set by the manufacturer (i.e. low range and normal range calibration curve). Each pooled plasma was tested three times in a single run. The slope and R2 of linear regression of mean of measured value and theoretical value, as well as bias, were calculated, and the linearity evaluation criterion was set according to National Health Standard of China and package inserts. (4) Limit of quantitation verification: The calibrator was diluted to two reference materials with activity values of 3.3% and 2.7%, and each material was tested twelve times. The limit of quantitation evaluation criterion was set according to CLSI document. Results: The CVs of intra-and inter-run were 1.0%-2.5% and 1.1%-2.6%, respectively. The biases of trueness verification were -0.4%, 1.0%, -2.6%, 0.3% and -2.7%, respectively. The linearity verification results of low range (3.6%-31.8%) and normal range (28.4%-160.4%) showed that the slopes of regression equation were 1.021 7 and 0.996 2, respectively, R2 were 0.993 5 and 0.993 9, respectively, and the biases with 0-1.8% and -10.1%-0 of plasmas met the criterion. The biases ranged from -0.4% to 0.3% of test results in the verification of limit of quantitation met the criterion. Conclusion: The verification results of VWF:GPIbM assay for precision, trueness, linearity and limit of quantification meet the performance requirements indicated in the package inserts and the criteria set in this study, which can be taken as a reference of performance verification for the clinical laboratories.

[Clinical application of the simultaneous detection of methotrexate and 7-hydroxymethotrexate in the delayed elimination for pediatric acute lymphoblastic leukemia].

Objective: To discuss the application value of the simultaneous determination of methotrexate (MTX) and 7-hydroxymethotrexate (7-OHMTX) in the delayed elimination of MTX for pediatric acute lymphoblastic leukemia (ALL). Methods: Cross sectional study. A total of 97 children who received 192 high-dose MTX treatments cycles in Lu Daopei Hospital from April to August 2019 were enrolled. The peripheral blood was collected at 0,24,48 h after the end of MTX infusion and analyzed by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). One hundred and ninety-two MTX treatments were divided into a normal MTX elimination group (n=149) and delayed elimination group (n=43) according to the standard of delayed elimination and divided into 0-9 year old group (n=95), 10-14 group (n=50), 15-18 group (n=47) according to age. The comparisons of the C(MTX), C(7-OHMTX) between normal and delayed group was conducted as well as among different age groups. Receiver operator characteristic curve (ROC) of C(MTX-0h) and C(7-OHMTX-0h) was analyzed and the concentration corresponding to the maximum of the Youden index on the ROC was set as the warning value for delayed elimination. Correlation between the delayed elimination after the end of MTX infusion and toxicity was investigated and the percentage of delayed elimination was also analyzed. Results: The concentrations of MTX and 7-OHMTX were significantly higher in the delayed elimination group than the normal group. Immediately after infusion (0 h), a C(7-OHMTX-0h) of >17.8 µmol/L (sensitivity 97.7%, specificity 54.4%) and a C(MTX-0h) of >148.8 µmol/L (sensitivity 72.1%, specificity 84.6%) were found to be warning predictors of delayed elimination under the MTX treatment protocol. MTX delayed elimination was positively correlated with methotrexate-induced toxicities (r=0.58, P<0.01). The percentage of hepatotoxicity and nephrotoxicity was 32.6% and 37.2% in the delayed elimination group, which was significantly higher than normal group of 12.8% and 3.4% (P<0.05). No significant difference was found in other toxicities. There was significant difference in C(MTX) among different age groups but no significant difference in C(7-OHMTX). Conclusion: Simultaneously determination of MTX and 7-OHMTX in plasma by HPLC-MS/MS in childhood ALL patients can provide a reference for clinical individualized medicine and pharmacokinetic research.

[Adult femur CT modeling and 3D automatic measurement of anatomical parameters].

Objective: To reconstruct a 3D model from adult femur CT scan data and automate a measurement of femoral anatomical parameters to study the characteristics of Chinese femur anatomical parameters. Methods: Using Mimics17.0, models from the CT data of 148 adult patients were established. The completed model was imported into Geomagic Studio and the anatomical landmarks of the femur were extracted to establish 3D coordinate system and unified coordinate system. Programmed with Matlab, using the nearest point iterative ICP algorithm and the 3D automatic extraction algorithm of anatomical landmarks to provide precision positioning, the femoral anatomical parameters were automatically measured and analyzed. The data were compared by using independent sample t test. Results: In this group, the diameter of the male femoral condyle was (84.1±3.6) mm, and it was (74.8±3.3) mm in the female; the anteroposterior diameter of the male femoral condyle was (66.5±3.7) mm, and it was (61.2±3.5) mm in the female; the diameter of the male ball head was (48.8±2.1) mm and it was (43.4±2.2) mm in the female; the differences between the two genders were all statistically significant (t=16.21, 8.84, 15.20, all P<0.05). The male femoral moment radius was (112.5±24.5) mm, and it was (124.7±19.2) mm in the female (t=3.30, P=0.002). The neck angle in male participants was 124.9°±4.0°, and it was 126.1°±5.5° in the female (t=1.40, P>0.05). As the height growed, most of the anatomical parameters increased accordingly. Conclusions: The 3D automatic measurement of femoral anatomical parameters is more reproducible and accurate than manual measurement. It is necessary to establish and enrich the femoral anatomical database to design and develop internal fixation products that meet the needs of Chinese people.

[Clinical observation on the anthracyclines-induced cardiotoxicity in patients with early-stage breast cancer].

Objective: To evaluate the anthracyclines-induced cardiotoxicity in patients with early-stage breast cancer. Methods: This retrospective study analyzed data of 64 patients (aged from 36 to 59 years old) with early-stage breast cancer after surgery. Patients were divided into ACT group (n=21), FAC group (n=19) and EC group (n=24). The NCI CTC 4.0 scores was used to evaluate the side effects at the time of 2 weeks, 4 weeks and 6 weeks after chemotherapy. Meanwhile, the level of cTnT, the incidence of abnormal electrocardiogram (ECG) and left ventricular ejection fraction (LVEF) were used to evaluate the anthracyclines-induced cardiotoxicity, the follow-up observation points were as follows: at the acute cardiotoxicity (time A), subacute cardiotoxicity (time B), 24 months after chemotherapy (time C), 36 months after chemotherapy (time D), 48 months after chemotherapy (time E), 60 months after chemotherapy (time F). The 3-years and 5-years overall survival and progress free disease survival among three groups were compared. Results: The ages, clinical stage, the size of tumor, axillary lymph node positivity and Eastern Cooperative Oncology Group Scores were similar among three groups (P>0.05); the incidence of side effects level 4 was 0. The levels of cTnT in the three groups were significantly lower than those at the baseline and time points C, D, E and F (all P<0.05), and the levels of cTnT were significantly higher in EC group than in FAC and ACT group at the time points B, C, D, E and F (P<0.05); however, the incidence of abnormal ECG and LVEF was similar among the 3 groups (P>0.05). The 5-year overall survival was 95.2% (20/21) ,100% (19/19) and 95.8% (23/24) in ACT group, FAC group and EC group, respectively; 5-year progress free disease survival was 95.2% (20/21) ,94.7% (18/19) and 91.7% (22/24) in ACT group, FAC group and EC group, respectively (P>0.05) . Conclusions: Patients with early-stage breast cancer after surgery could tolerate the anthracyclines-induced cardiotoxicity. Three chemotherapy schemes of ACT, FAC and EC, especially the EC protocol, could affect the myocardial damage. However, outcome is comparable among patients treated with above chemotherapy schemes in this patient cohort.

[Sarcopenia and long-term prognosis of patients with chronic heart failure in patients aged 70 years and over].

To explore the association between sarcopenia (SAR) and long-term prognosis of patients with chronic heart failure (CHF) aged 70 years and over, 182 CHF patients from January 1, 2012 to December 31, 2014 were included in the present study. The patients were divided into the SAR group and the non-SAR group. The median follow-up period was 36 (3, 57) months. The endpoint was any heart failure-related event (HFRE). There were significant differences in age, body mass index, hemoglobin, B-type natriuretic peptide, hypersensitive troponin T (hs-TnT), left ventricular ejection fraction (LVEF) and cardiac function class between the two groups (all P<0.05). The Kaplan-Meier analysis showed that the survival time of the non-SAR patients was much longer than that of the SAR patients (P<0.05). The multivariate Cox regression analysis indicated that SAR was an independent risk factor for HFRE, suggesting a role of sarcopenia on long-term prognosis of patients with chronic heart failure.

CDK6-mediated repression of CD25 is required for induction and maintenance of Notch1-induced T-cell acute lymphoblastic leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subset of acute leukemia, characterized by frequent activation of Notch1 or AKT signaling, where new therapeutic approaches are needed. We showed previously that cyclin-dependent kinase 6 (CDK6) is required for thymic lymphoblastic lymphoma induced by activated AKT. Here, we show CDK6 is required for initiation and maintenance of Notch-induced T-ALL. In a mouse retroviral model, hematopoietic stem/progenitor cells lacking CDK6 protein or expressing kinase-inactive (K43M) CDK6 are resistant to induction of T-ALL by activated Notch, whereas those expressing INK4-insensitive (R31C) CDK6 are permissive. Pharmacologic inhibition of CDK6 kinase induces CD25 and RUNX1 expression, cell cycle arrest and apoptosis in mouse and human T-ALL. Ablation of Cd25 in a K43M background restores Notch-induced T leukemogenesis, with disease that is resistant to CDK6 inhibitors in vivo. These data support a model whereby CDK6-mediated suppression of CD25 is required for initiation of T-ALL by activated Notch1, and CD25 induction mediates the therapeutic response to CDK6 inhibition in established T-ALL. These results both validate CDK6 as a molecular target for therapy of this subset of T-ALL and suggest that CD25 expression could serve as a biomarker for responsiveness of T-ALL to CDK4/6 inhibitor therapy.

Amyloidogenic variant of apolipoprotein A-I elicits cellular stress by attenuating the protective activity of angiogenin.

Amyloidogenic 'gain-of-function' mutations in apolipoprotein A-I (ApoA-I) gene (APOA1) result in systemic amyloidosis characterized by aggregate deposition and eventually cell death. However, how amyloidogenic variants of ApoA-I induce cell death is unknown. Here we report that one of the mechanisms by which amyloidogenic ApoA-I induces cell death is through attenuating anti-stress activity of angiogenin (ANG), a homeostatic protein having both pro-growth and pro-survival functions. Under growth conditions, ANG is located in nucleolus where it promotes ribosomal RNA (rRNA) transcription thereby stimulating cell growth. In adverse conditions, ANG is relocated to cytoplasm to promote damage repairs and cell survival. We find that in cells overexpressing the L75P-APOA1 mutant ANG expression is decreased and normal cellular localization of ANG is altered in response to stress and growth signals. In particular, ANG does not relocate to cytoplasm under stress conditions but is rather retained in the nucleolus where it continues promoting rRNA transcription, thus imposing a ribotoxic effect while simultaneously compromising its pro-survival activity. Consistently, we also find that addition of exogenous ANG protects cells from L75P-ApoA-I-induced apoptosis.

Effect of waterborne copper exposure on growth, hepatic enzymatic activities and histology in Synechogobius hasta.

The present study was conducted to determine growth, hepatic enzymatic activities and histology in Synechogobius hasta exposed to waterborne copper concentrations of 0 (control), 0.15 and 0.3 mg Cu/l, respectively, for 15 days, and explore whether waterborne copper exposure could induce the fatty liver syndrome for the fish species. Growth (WG and SGR) declined, but HSI increased in S. hasta with increasing waterborne copper levels (P<0.05). Waterborne copper exposure also significantly increased lipid content and reduced protein content in both whole body and liver, and increased copper accumulation in whole body and vertebrae. Copper exposure changed hepatic enzymatic activities (SOD, CAT, SDH, PK, LDH, LPL and HL) and increased hepatic lipid peroxidation level, impaired the histological structure of the gill and liver in S. hasta. Thus, our study demonstrated for the first time that waterborne Cu exposure could induce fatty liver syndrome in fish.

Inactivation of AMPK alters gene expression and promotes growth of prostate cancer cells.

AMP-activated protein kinase (AMPK) serves as a fuel-sensing enzyme that is activated by binding of AMP and subsequent phophorylation by upstream kinases such as the tumor suppressor LKB1, when cells sense an increase in the ratio of AMP to ATP. Acute activation of AMPK stimulates fatty acid oxidation to generate more ATP and simultaneously inhibits ATP-consuming processes including fatty acid and protein syntheses, thereby preserving energy for acute cell-surviving program, whereas chronic activation leads to inhibition of cell growth. The goal of the present study is to explore the mechanisms by which AMPK regulates cell growth. Toward this end, we established stable cell lines by introducing a dominant-negative mutant of AMPK alpha1 subunit or its shRNA into the prostate cancer C4-2 cells and other cells, or wild type LKB1 into the lung adenocarcinoma A549 and breast MB-MDA-231 cancer cells, both of which lack functional LKB1. Our results showed that the inhibition of AMPK accelerated cell proliferation and promoted malignant behavior such as increased cell migration and anchorage-independent growth. This was associated with decreased G1 population, downregulation of p53 and p21, and upregulation of S6K, IGF-1 and IGF1R. Conversely, treatment of the C4-2 cells with 5-aminoimidazole-4-carboxamide 1-D-ribonucleoside (AICAR), a prototypical AMPK activator, caused opposite changes. In addition, our study using microarray and RT-PCR revealed that AMPK regulated gene expression involved in tumor cell growth and survival. Thus, our study provides novel insights into the mechanisms of AMPK action in cancer cells and presents AMPK as an ideal drug target for cancer therapy.

Gene profiling after knocking-down expression of nucleostemin in Hela cells using oligonucleotide DNA microarray.

Nucleostemin (NS) is preferentially and exclusively expressed in the stem cells and cancer cells, but not in differentiated adult tissues and cells. NS is likely to take part in controlling the proliferation and differentiation switch in stem cells and progenitor cells. Its deregulation in cancer also contributes to the elevated proliferation and undifferentiation of cancer cells. However, the mechanisms by which NS helps to maintain both cancer and stem cells in undifferentiated state remain unclear. In this study, we carried out gene profilings using oligonucleotide DNA microarray after knocking down the expression of NS in Hela cells. Of the 21,329 genes, 200 genes were found differentially expressed in NS silenced Hela cells with > 2 fold ratio (either > 2 or < 0.5). Category analysis indicated these differential genes were mainly related with cancer pathogenesis, cell death, cell growth and proliferation. NS related gene pathway analysis suggested NS was mostly involved in the networks of cell cycle and differentiation controls. p53 may not be the only partner of NS in its regulated pathways. c-Myc may directly or indirectly interact with it to control the proliferation and differentiation switch in cancer cells. Our study provides a general view of the NS-target genes, and indicates the possible pathways in which NS plays its role in proliferation control.

Complete 1H and 13C assignments of 8-C-beta-D-[2-O-(E)-p-coumaroyl]glucopyranosyl-2-(2-hydroxy)propyl-7-methoxy-5-methylchromone.

1H and 13C NMR spectra of 8-C-beta-D-[2-O-(E)-p-coumaroyl] glucopyranosyl-2-(2-hydroxy)propyl-7-methoxy-5-methylchromone were completely assigned by 2D NMR observations. Especially the 1H assignments of the glucosyl and hydroxyl protons were achieved by utilizing HMQC, HMBC, 1H-1H COSY and DEPT techniques together with a heavy water exchange 1H NMR experiment.

Angiogenin activates Erk1/2 in human umbilical vein endothelial cells.

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.

Neomycin inhibits the angiogenic activity of fibroblast and epidermal growth factors.

Neomycin has been shown to block nuclear translocation of angiogenin in endothelial cells, thereby inhibiting its capacity to induce angiogenesis. We report here that neomycin is also an effective inhibitor of angiogenesis induced by acidic fibroblast growth factor, basic fibroblast growth factor, and epidermal growth factor, all of which undergo nuclear translocation, but not that of vascular endothelial growth factor which does not undergo nuclear translocation. Blocking nuclear translocation, therefore, seems to be a general mechanism for the antiangiogenesis action of neomycin applicable to those angiogenic factors that require nuclear translocation for angiogenesis. These results along with the known toxicity and pharmacokinetics profiles make neomycin and its analogues good candidate inhibitors of angiogenesis that might be developed as antitumor agents.

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development. Oncogene (2001) 20, 24 - 33.

Expression of liver cancer associated gene HCCA3.

AIM: To study and clone a novel liver cancer related gene, and to explore the molecular basis of liver cancer genesis. METHODS: Using mRNA differential display polymerase chain reaction (DDPCR), we investigated the difference of mRNA in human hepatocellular carcinoma (HCC) and paired surrounding liver tissues, and got a gene probe. By screening a human placenta cDNA library and genomic homologous extend, we obtained a full-length cDNA named HCCA3. We analyzed the expression of this novel gene in 42 pairs of HCC and the surrounding liver tissues, and distribution in human normal tissues by means of Northern blot assay. RESULTS: A full-length cDNA of liver cancer associated gene HCCA3 has been submitted to the GeneBank nucleotide sequence databases (Accession No. AF276707). The positive expression rate of this gene was 78.6% (33/42) in HCC tissues, and the clinical pathological data showed that the HCCA3 was closely associated with the invasion of tumor capsule (P=0.023) and adjacant small metastasis satellite nodules lesions (P=0.041). The HCCA3 was widely distributed in the human normal tissues, which was intensively expressed in lungs, brain and colon tissues, while lowly expressed in the liver tissues. CONCLUSION: A novel full-length cDNA was cloned and differentiated, which was highly expressed in liver cancer tissues. The high expression was closely related to the tumor invasiveness and metastasis,that may be the late heredited change in HCC genesis.

Fibroblast growth factors are translocated to the nucleus of human endothelial cells in a microtubule- and lysosome-independent pathway.

Exogenous acidic and basic fibroblast growth factors undergo rapid nuclear translocation in human umbilical vein endothelial cells. When nuclear translocation reaches saturation, more than 70% of the internalized growth factors are in the nuclear fraction. Lysosomal inhibitors, such as leupeptin and chloroquine, and microtubule inhibitors including colchicine and 2-methoxyl-beta-estradiol neither increase nor decrease nuclear translocation. The results suggest that nuclear translocation of fibroblast growth factors does not require cytosolic accumulation or lysosomal processing and that the transportation of exogenous growth factors across the cytoplasm is independent of microtubules.

Human angiogenin is rapidly translocated to the nucleus of human umbilical vein endothelial cells and binds to DNA.

Human angiogenin is translocated to the nucleus of human umbilical vein endothelial cells in a time-dependent manner. Exogenous angiogenin appears in the nucleus in 2 min, reaches saturation in 15 min when 85% of the internalized angiogenin is in the nuclei, and remains associated with the nucleus for at least 4 h. Endothelial cells cultured at low density have a much higher capacity to translocate angiogenin to the nucleus than do those cultured at high density. This observation is consistent with previous findings that both the ability of endothelial cells to proliferate in response to angiogenin and the expression of an angiogenin receptor on the cell surface depend on cell density. Nuclear (125)I-angiogenin is not degraded and is neither spontaneously dissociated nor replaced by unlabeled angiogenin. It is, however, released by deoxyribonuclease I, but not by ribonuclease A, suggesting that angiogenin binds to DNA in the nucleus. These results suggest that in addition to acting as a ribonuclease, angiogenin may play a role in regulating gene expression by direct binding to DNA.

Neomycin inhibits angiogenin-induced angiogenesis.

A class of angiogenesis inhibitor has emerged from our mechanistic study of the action of angiogenin, a potent angiogenic factor. Neomycin, an aminoglycoside antibiotic, inhibits nuclear translocation of human angiogenin in human endothelial cells, an essential step for angiogenin-induced angiogenesis. The phospholipase C-inhibiting activity of neomycin appears to be involved, because U-73122, another phospholipase C inhibitor, has a similar effect. In contrast, genistein, oxophenylarsine, and staurosporine, inhibitors of tyrosine kinase, phosphotyrosine phosphatase, and protein kinase C, respectively, do not inhibit nuclear translocation of angiogenin. Neomycin inhibits angiogenin-induced proliferation of human endothelial cells in a dose-dependent manner. At 50 microM, neomycin abolishes angiogenin-induced proliferation but does not affect the basal level of proliferation and cell viability. Other aminoglycoside antibiotics, including gentamicin, streptomycin, kanamycin, amikacin, and paromomycin, have no effect on angiogenin-induced cell proliferation. Most importantly, neomycin completely inhibits angiogenin-induced angiogenesis in the chicken chorioallantoic membrane at a dose as low as 20 ng per egg. These results suggest that neomycin and its analogs are a class of agents that may be developed for anti-angiogenin therapy.

Inhibition of human angiogenin by DNA aptamers: nuclear colocalization of an angiogenin-inhibitor complex.

Specific ligands (aptamers) for angiogenin were selected from a 72-mer oligodeoxynucleotide library consisting of 28 randomized positions flanked by two constant regions of 22 residues each. From a starting pool of approximately 10(14) molecules, 19 angiogenin-binding ligands were obtained. Among them, two oligonucleotides showed significant inhibition of the ribonucleolytic activity of angiogenin with apparent Kis of 0.65 and 0.60 micro M, respectively. One of them was shortened on the basis of its secondary structure to provide a 45-mer oligonucleotide that retained much of the inhibitory properties of the parent molecule. It inhibits both the angiogenic and cell proliferative activities of angiogenin but does not interfere with its nuclear translocation in human endothelial cells. Importantly, the inhibitor is cotranslocated to the nucleus with angiogenin in a approximately 1:1 stoichiometric ratio. These results demonstrate that the inhibition of angiogenin-induced cell proliferation and angiogenesis by the oligonucleotide is due to suppression of the ribonucleolytic activity of angiogenin, an event that occurs most likely within the cell nucleus.