OBJECTIVE: MicroRNA-155 (miR-155) is significantly highly expressed in breast cancer, lung cancer, liver cancer and other malignant tumors. This study was to design and construct a radiolabeled probe targeting miR-155 for in vivo imaging in breast cancer. METHODS: Anti-miR-155 oligonucleotide (AMO-155) was chemically synthesized with 2' OMe modification. Its 5' end was linked with acetyl amine group. After chelated with a bifunctional chelator NHS-MAG3, AMO-155 was radiolabeled with ^99mTc using stannous chloride. The serum stability was evaluated at cellular level. In vivo imaging was performed in MCF-7 tumor bearing mice after the administration of ^99mTc radiolabeled AMO-155 and scramble control probes, respectively. Furthermore, the blocked imaging of tumor bearing mice was obtained after the injection of unlabeled AMO-155 2 hours ahead. MCF-7 and MDA-MB-231 tumor bearing mice with different expression level of miR-155 were imaged, respectively. Quantitative real-time PCR (qRT-PCR) was used to identify the expression level of miR-155 in the bearing tumors. RESULTS: ^99mTc-AMO-155 was prepared with high radiolabeled efficiency (97%), radiochemical purity (greater than 98%), and radioactive specific activity (3.75 GBq/μg). ^99mTc-AMO-155 was stable in fresh human serum for 12 hours. After the administration via tail vein, ^99mTc-AMO-155 displayed significant accumulation in MCF-7 bearing tumors with high expression level of miR-155, whereas ^99mTc-control showed little accumulation. After blocked with unlabeled AMO-155, the tumor could not be visualized clearly after the administration of ^99mTc-AMO-155. Furthermore, ^99mTc-AMO-155 could show the differential expression of miR-155 in vivo. MCF-7 tumor was shown with significantly higher radioactive accumulation than MDA-MB-231, based on its higher expression level of miR-155, which was verified by qRT-PCR. CONCLUSION ^99mTc-labeled AMO-155 with chemical modification showed good serum stability and in vivo tumor targeting ability. This study provides a potential probe for in vivo imaging of breast cancer.
Animals ; Breast Neoplasms/diagnostic imaging* ; Cell Line, Tumor ; Female ; Humans ; Mice ; MicroRNAs/analysis* ; Oligonucleotides, Antisense ; Oligopeptides ; Radiopharmaceuticals ; Succinimides ; Technetium ; Tissue Distribution

Aplastic anemia may develop secondary to environmental exposure to entities such as chemicals, medical drugs, and infectious agents. Fatal complications from antiepileptic medications may occur despite careful and appropriate use. We report the case of a 9-year-old girl with a presenting diagnosis of aplastic anemia following treatment with ethosuximide for absence seizures. Aplastic anemia can now be cured with stem cell transplantation or immunosuppressive therapy. In this case, however, because of the impossibility of bone marrow transplantation and the specific needs of the patient's parents, three courses of methylprednisolone pulse therapy were administered. Following the therapy, there was improvement in pancytopenia and complete remission in the bone marrow. No adverse side effects of therapy were observed. The authors suggest that methylprednisolone pulse therapy may be a treatment for acquired aplastic anemia.
Anemia, Aplastic* ; Anticonvulsants ; Bone Marrow ; Bone Marrow Transplantation ; Child ; Diagnosis ; Environmental Exposure ; Epilepsy, Absence ; Ethosuximide ; Female ; Humans ; Methylprednisolone* ; Pancytopenia ; Parents ; Stem Cell Transplantation

The ubiquitin-proteasome system plays a pivotal role in breast tumorigenesis by controlling transcription factors, thus promoting cell cycle growth, and degradation of tumor suppressor proteins. However, breast cancer patients have failed to benefit from proteasome inhibitor treatment partially due to proteasome heterogeneity, which is poorly understood in malignant breast neoplasm. Chemical crosslinking is an increasingly important tool for mapping protein three-dimensional structures and proteinprotein interactions. In the present study, two cross-linkers, bis (sulfosuccinimidyl) suberate (BS(3)) and its water-insoluble analog disuccinimidyl suberate (DSS), were used to map the subunit-subunit interactions in 20S proteasome core particle (CP) from MDA-MB-231 cells. Different types of gel electrophoresis technologies were used. In combination with chemical cross-linking and mass spectrometry, we applied these gel electrophoresis technologies to the study of the noncovalent interactions among 20S proteasome subunits. Firstly, the CP subunit isoforms were profiled. Subsequently, using native/SDSPAGE, it was observed that 0.5 mmol/L BS(3) was a relatively optimal cross-linking concentration for CP subunit-subunit interaction study. 2-DE analysis of the cross-linked CP revealed that α1 might preinteract with α2, and α3 might pre-interact with α4. Moreover, there were different subtypes of α1α2 and α3α4 due to proteasome heterogeneity. There was no significant difference in cross-linking pattern for CP subunits between BS(3) and DSS. Taken together, the gel-based characterization in combination with chemical cross-linking could serve as a tool for the study of subunit interactions within a multi-subunit protein complex. The heterogeneity of 20S proteasome subunit observed in breast cancer cells may provide some key information for proteasome inhibition strategy.
Amino Acid Sequence ; Breast Neoplasms ; drug therapy ; genetics ; pathology ; Cell Line, Tumor ; Cross-Linking Reagents ; administration & dosage ; Electrophoresis, Gel, Two-Dimensional ; Female ; Humans ; Mass Spectrometry ; Proteasome Endopeptidase Complex ; drug effects ; Protein Binding ; drug effects ; Protein Isoforms ; genetics ; Protein Subunits ; biosynthesis ; genetics ; Proteomics ; Succinimides ; administration & dosage

BACKGROUND AND PURPOSE: Childhood absence epilepsy (CAE) is one of the most common types of pediatric epilepsy. It is generally treated with ethosuximide (ESM), valproic acid (VPA), or lamotrigine (LTG), but the efficacy and adverse effects of these drugs remain controversial. This study compared initial therapy treatment outcomes, including VPA-LTG combination, and assessed clinical factors that may predict treatment response and prognosis. METHODS: Sixty-seven patients with typical CAE were retrospectively enrolled at the Korea University Medical Center. We reviewed patients' clinical characteristics, including age of seizure onset, seizure-free interval, duration of seizure-free period, freedom from treatment failure, breakthrough seizures frequency, and electroencephalogram (EEG) findings. RESULTS: The age at seizure onset was 7.9±2.7 years (mean±SD), and follow-up duration was 4.4±3.7 years. Initially, 22 children were treated with ESM (32.8%), 23 with VPA (34.3%), 14 with LTG (20.9%), and 8 with VPA-LTG combination (11.9%). After 48 months of therapy, the rate of freedom from treatment failure was significantly higher for the VPA-LTG combination therapy than in the three monotherapy groups (p=0.012). The treatment dose administrated in the VPA-LTG combination group was less than that in the VPA and LTG monotherapy groups. The shorter interval to loss of 3-Hz spike-and-wave complexes and the presence of occipital intermittent rhythmic delta activity on EEG were significant factors predicting good treatment response. CONCLUSIONS: This study showed that low-dose VPA-LTG combination therapy has a good efficacy and fewer side effects than other treatments, and it should thus be considered as a firstline therapy in absence epilepsy.
Academic Medical Centers ; Child ; Electroencephalography ; Epilepsy ; Epilepsy, Absence* ; Ethosuximide ; Follow-Up Studies ; Freedom ; Humans ; Korea ; Prognosis ; Retrospective Studies ; Seizures ; Treatment Failure ; Valproic Acid

OBJECTIVETo investigate the ability of Porphyromonas gingivalis to invade human periodontal ligament cells (hPDLCs) and the effect of intracellular P. gingivalis on cell proliferation and osteogenic differentiation in vitro.

METHODSThe invasion ability of P. gingivalis in hPDLCs was tested using an antibiotic protection assay at the multiplicity of infection (MOI) of 10 and 100. The proliferation of the infected cells was detected using a CFDA-SE kit, and the cells were sorted by fluorescence-activated cell sorting (FACS) followed by alizarin red staining for detecting mineralization nodules deposition; real-time PCR was used to examine the expression of Runx2 mRNA in the cells.

RESULTSP. gingivalis actively invaded hPDLCs, and the internalized P. gingivalis was able to resist antibiotic treatment. The cells infected by P. gingivalis exhibited no significant suppression of cell proliferation, but showed significantly lowered capacity for osteogenic differentiation, down-regulated RUNX2 mRNA expression, and reduced mineral deposition.

CONCLUSIONIntracellular P. gingivalis does not significantly affect the proliferation of hPDLCs but inhibits osteogenic differentiation of the cells.

Cell Differentiation ; Cell Proliferation ; Cells, Cultured ; Core Binding Factor Alpha 1 Subunit ; metabolism ; Flow Cytometry ; Fluoresceins ; Humans ; Osteogenesis ; Periodontal Ligament ; cytology ; microbiology ; Porphyromonas gingivalis ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Succinimides

OBJECTIVETo test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro.

METHODSThe anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined.

RESULTSThe labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%.

CONCLUSIONThe (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.

Humans ; Isotope Labeling ; Liposomes ; MicroRNAs ; genetics ; Myocytes, Cardiac ; Oligonucleotides ; Oligonucleotides, Antisense ; Oligopeptides ; Radiopharmaceuticals ; Silicon Dioxide ; Succinimides ; Transfection

Cross-talk between the thalamus and cortex has been implicated in attention but its pathogenic role in attention-deficit/hyperactivity disorder (ADHD) remains unknown. Here, I demonstrate that Git1-/- mice, previously proposed as an animal model for ADHD, show abnormal theta oscillation in the thalamus. Multi-electrode recordings revealed that Git1-/- mice have hyper-synchrony of neural activities between the thalamus and cortex. The abnormal thalamic oscillation and thalamocortical synchrony in Git1-/- mice were markedly reduced by amphetamine. In addition, ethosuximide ameliorates abnormal thalamic oscillation and ADHD-like hyperactivity shown in Git1-/- mice. My study suggests critical roles of GIT1 and thalamocortical neural circuitry in ADHD.
Amphetamine ; Animals ; Ethosuximide ; Mice* ; Models, Animal ; Thalamus

OBJECTIVETo explore the feasibility and fluorescence characteristics of CFSE negative staining for in vivo cell imaging of super paramagnetic iron oxide nanoparticles (SPIO) phagocytosed by mouse mononuclear macrophage leukemia cells-RAW264.7.

METHODSAfter labeled with SPIO, the RAW264.7 macrophages were stained with Prussian blue stain and CFSE fluorescence negative stain step by step. Furthermore, trypan blue staining was used to evaluate cell viability of cells which stained with CFSE. At last, laser scanning confocal microscope was used to measure SPIO in cells through CFSE fluorescence negative stain method.

RESULTSSPIO within RAW264.7 macrophages showed blue in Prussian's blue staining, while showed negative area in CFSE negative staining. Good consistencies between Prussian's blue staining and CFSE negative staining were observed. In addition, RAW264.7 macrophages showed high viability after SPIO/CFSE dual-labeled method, proved by typan stain.

CONCLUSIONThe CFSE fluorescence negative staining may be used for detecting SPIO that phagocytosed by RAW264.7 macrophages and it is showed good consistency that confirmed one another when compared to classic Prussian' blue staining.

Animals ; Cell Line, Tumor ; Cell Survival ; Contrast Media ; Ferric Compounds ; Ferrocyanides ; Fluoresceins ; Fluorescence ; Leukemia ; Macrophages ; Magnetic Resonance Imaging ; Magnetite Nanoparticles ; Mice ; Negative Staining ; Phagocytosis ; Succinimides

PURPOSE: Ethosuximide (ESX) is currently not available due to various reasons in Korea. The aim of this study is to compare the efficacy of valproate (VPA) and lamotrigine (LTG) when ESX monotherapy was replaced by VPA or LTG. METHODS: A retrospective study was done for a total of 34 patients treated with ESX in 5 different hospitals affiliated with Catholic University of Korea from January, 2010 to December, 2012. They all were initially treated with ESX, but later switched to VPA or LTG. The subjects were selected based on clinical symptoms and electroencephalography findings. RESULTS: Among 34 patients, VPA was prescribed to 17 patients (50.0%) and LTG to 17 patients (50.0%). Twenty patients (58.8%) achieved the seizure freedom after 3 months of the treatments, 13 patients (76.5%) by VPA and 7 (41.2%) by LTG respectively. Four patients (23.5%) with VPA and 10 (58.8%) with LTG were replaced by other anticonvulsants due to ineffectiveness and/or side effects of medication. When we compare the efficacy of seizure reduction between VPA and LTG after 3 month period of the treatment, the efficacy of VPA was better than that of LTG (P=0.04). CONCLUSION: The results of this study suggest that the VPA is a better alternative anticonvulsant than LTG for the patients with absence epilepsy who are unable to continue ESX.
Anticonvulsants ; Child ; Electroencephalography ; Epilepsy ; Epilepsy, Absence* ; Ethosuximide ; Freedom ; Humans ; Korea ; Retrospective Studies ; Seizures ; Valproic Acid*

OBJECTIVE: Authors aimed to determine the targeting ability of vascular endothelial growth factor receptor 2 (VEGFR2)-conjugated quantum dots (QDs) in vitro, and apply it for a xenograft prostate cancer mouse model. MATERIALS AND METHODS: Conjugation reaction of QDs was performed by using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and sulfo-(N-hydroxysulfosuccinimide) (Sulfo-NHS). The human umbilical vein cord endothelial cells (HUVECs) were incubated with QDs, conjugated with antiVGFR2, to see a specific binding in vitro. Fluorescent cell images were taken by a confocal microscope. The human prostate cancer cells (PC3) were injected to five nude mice on hind limbs to make the xenograft tumor model. QD-antiVEGFR2 antibody complex was injected into the tumor model and fluorescence measurements were performed at 1, 4, 9, 12, 15, and 24 hours after the injection. RESULTS: The specific interaction between HUVECs and QD-antiVEGFR2 antibody was clearly shown in vitro. The in vivo fluorescence image disclosed that there was an increased signal of tumor, 12 hours after the injection of QDs. CONCLUSION: By showing endothelial cells binding with QDs-antiVEGFR2 antibodyand an experimental application of the antibody for VEGFR2 imaging in the prostate cancer xenograft mouse model, we suggests that the antibody-conjugated QDs can be a potential imaging tool for angiogenesis of the cancer.
Animals ; Carbodiimides/pharmacology ; Cell Line, Tumor ; Disease Models, Animal ; Electrophoresis, Agar Gel ; Fluorescence ; Male ; Mice ; Mice, Nude ; Microscopy, Confocal ; Neovascularization, Pathologic/*pathology ; Prostatic Neoplasms/*pathology ; *Quantum Dots ; Succinimides/pharmacology ; Transplantation, Heterologous ; Vascular Endothelial Growth Factor Receptor-2/*antagonists & inhibitors