PURPOSE: Ovarian cancer (OC) is the most fatal of gynecological malignancies with a high rate of recurrence. We aimed to evaluate the expression of solute carrier family 6, member 12 (SLC6A12) and methylation of its promoter CpG sites in a xenograft mouse model of metastatic OC, and to investigate the regulatory mechanisms that promote aggressive properties during OC progression. MATERIALS AND METHODS: Expression of SLC6A12 mRNA was determined by reverse-transcription quantitative polymerase chain reaction (RT-qPCR), and DNA methylation status of its promoter CpGs was detected by quantitative methylation-specific PCR. The metastatic potential of SLC6A12 was evaluated by in vitro migration/invasion transwell assays. Gene expression and DNA methylation of SLC6A12 and clinical outcomes were further investigated from publicly available databases from curatedOvarianData and The Cancer Genome Atlas. RESULTS: SLC6A12 expression was 8.1–14.0-fold upregulated and its DNA methylation of promoter CpG sites was 41–62% decreased in tumor metastases. After treatment with DNA methyltransferase inhibitor and/or histone deacetylase inhibitor, the expression of SLC6A12 was profoundly enhanced (~8.0-fold), strongly supporting DNA methylation-dependent epigenetic regulation of SLC6A12. Overexpression of SLC6A12 led to increased migration and invasion of ovarian carcinoma cells in vitro, approximately 2.0-fold and 3.3-fold, respectively. The meta-analysis showed that high expression of SLC6A12 was significantly associated with poor overall survival [hazard ratio (HR)=1.07, p value=0.016] and that low DNA methylation levels of SLC6A12 at specific promoter CpG site negatively affected patient survival. CONCLUSION: Our findings provide novel evidence for the biological and clinical significance of SLC6A12 as a metastasis-promoting gene.
Animals ; Carrier Proteins/genetics/*metabolism ; Cell Line, Tumor ; Cell Migration Assays ; *CpG Islands ; *DNA Methylation ; Disease Progression ; Epigenesis, Genetic ; Female ; Gene Expression Regulation, Neoplastic ; Humans ; Mice ; Neoplasm Invasiveness ; Neoplasm Transplantation ; Ovarian Neoplasms/genetics/*metabolism/mortality/pathology ; Polymerase Chain Reaction ; Prognosis ; *Promoter Regions, Genetic ; RNA, Messenger/*metabolism ; Up-Regulation

In the present study, we used a proteomics approach based on a two-dimensional electrophoresis (2-DE) reference map to investigate protein expression in the ovarian tissues of pubertal Swiss-Webster mice subjected to carbon ion radiation (CIR). Among the identified proteins, ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) is associated with the cell cycle[1] and that it influences proliferation in ovarian tissues. We analyzed the expression of UCH-L1 and the proliferation marker proliferation cell nuclear antigen (PCNA) following CIR using immunoblotting and immunofluorescence. The proteomics and biochemical results provide insight into the underlying mechanisms of CIR toxicity in ovarian tissues.
Animals ; Biomarkers ; Carrier Proteins ; genetics ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; Female ; Gene Expression ; Heavy Ion Radiotherapy ; adverse effects ; Mice ; Ovary ; radiation effects ; Proteomics ; Random Allocation ; Ubiquitin Thiolesterase ; genetics ; metabolism

OBJECTIVETo investigate the mutations of SLC22A5 gene in patients with systemic primary carnitine deficiency (CDSP).

METHODSHigh liquid chromatography tandem mass spectrometry (HPLC/MS/MS) was applied to screen congenital genetic metabolic disease and eight patients with CDSP were diagnosed among 77 511 samples. The SLC22A5 gene mutation was detected using massarray technology and sanger sequencing. Using SIFT and PolyPhen-2 to predict the function of protein for novel variations.

RESULTSTotal detection rate of gene mutation is 100% in the eight patients with CDSP. Seven patients had compound heterozygous mutations and one patient had homozygous mutations. Six different mutations were identified, including one nonsense mutation [c.760C>T(p.R254X)] and five missense mutations[c.51C>G(p.F17L), c.250T>A(p.Y84N), c.1195C>T(p.R399W), c.1196G>A(p.R399Q), c.1400C>G(p.S467C)]. The c.250T>A(p.Y84N) was a novel variation, the novel variation was predicted to have affected protein structure and function. The c.760C>T (p.R254X)was the most frequently seen mutation, which was followed by the c.1400C>G(p.S467C).

CONCLUSIONThis study confirmed the diagnosis of eight patients with CDSP on the gene level. Six mutations were found in the SLC22A5 gene, including one novel mutation which expanded the mutational spectrum of the SLC22A5 gene.

Adult ; Amino Acid Sequence ; Base Sequence ; Cardiomyopathies ; diagnosis ; genetics ; metabolism ; Carnitine ; deficiency ; genetics ; metabolism ; DNA Mutational Analysis ; methods ; Female ; Gene Frequency ; Genotype ; Humans ; Hyperammonemia ; diagnosis ; genetics ; metabolism ; Infant, Newborn ; Male ; Muscular Diseases ; diagnosis ; genetics ; metabolism ; Mutation ; Organic Cation Transport Proteins ; genetics ; metabolism ; Reproducibility of Results ; Sensitivity and Specificity ; Sequence Homology, Amino Acid ; Solute Carrier Family 22 Member 5 ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

OBJECTIVETo identify pathogenic mutations in a Chinese pedigree affected with methylmalonic academia for genetic counseling and prenatal diagnosis.

METHODSMolecular analysis of the MUT, MMACHC, MMAA and MMAB genes was performed for the proband with methylmalonic academia by Ion Torrent semiconductor sequencing. Candidate mutations were validated by Sanger sequencing. The couple was offered prenatal diagnosis via analyzing of the fetal DNA through amniocentesis.

RESULTSThe proband was found to be compound heterozygous for c.609G>A (p.Trp203X) and c.658-660del AAG (p.Lys220del) mutations, which were inherited respectively from each of his parents. Prenatal diagnosis showed that the fetus has inherited two wild-type parental alleles.

CONCLUSIONThe targeted Ion Torrent PGM sequencing has detected pathogenic mutations in the Chinese pedigree affected with methylmalonic academia, which has provided molecular evidence for clinical diagnosis, genetic counseling and prenatal diagnosis for the family.

Adult ; Alkyl and Aryl Transferases ; genetics ; Amino Acid Metabolism, Inborn Errors ; embryology ; genetics ; Asian Continental Ancestry Group ; genetics ; Base Sequence ; Carrier Proteins ; genetics ; China ; Female ; High-Throughput Nucleotide Sequencing ; instrumentation ; methods ; Humans ; Infant ; Male ; Methylmalonyl-CoA Mutase ; genetics ; Mitochondrial Membrane Transport Proteins ; genetics ; Molecular Sequence Data ; Mutation ; Pedigree ; Pregnancy ; Prenatal Diagnosis ; instrumentation ; methods

Gephyrin is a central element that anchors, clusters and stabilizes glycine and gamma-aminobutyric acid type A receptors at inhibitory synapses of the mammalian brain. It self-assembles into a hexagonal lattice and interacts with various inhibitory synaptic proteins. Intriguingly, the clustering of gephyrin, which is regulated by multiple posttranslational modifications, is critical for inhibitory synapse formation and function. In this review, we summarize the basic properties of gephyrin and describe recent findings regarding its roles in inhibitory synapse formation, function and plasticity. We will also discuss the implications for the pathophysiology of brain disorders and raise the remaining open questions in this field.
Animals ; Carrier Proteins/chemistry/genetics/*metabolism ; Disease Susceptibility ; GABAergic Neurons/*metabolism ; Gene Expression Regulation ; Humans ; Membrane Proteins/chemistry/genetics/*metabolism ; Protein Binding ; Protein Interaction Domains and Motifs ; Protein Processing, Post-Translational ; Protein Transport ; Synapses/*metabolism

With the development of the quality of life, the morbidity of hyperuricemia is increasing year by year. At the same time, it appears that this disease attacks the young people currently. As the study of pathogenesis of hyperuricemia advanced, a series of uric acid transporters were found during this process. Meanwhile, the definition of transporterome was proposed. They were divided into three groups according to the functions: reabsorption proteins, excretion proteins and skeleton proteins. At moment, the drugs for hyperuricmia mainly include uric acid composition inhibitors and uric acid excretion promoters. Since the excretion of uric acid plays a leading role during the process of attack of hyperurecimia, it makes sense to explore Chinese medicines with clear mechanism targeting the transporterome. Therefore, this paper would focus on transporterome and summarize the mechanisms of Chinese medicines in treating hyperuricemia.
Animals ; Biological Transport ; Carrier Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Drugs, Chinese Herbal ; therapeutic use ; Humans ; Hyperuricemia ; drug therapy ; metabolism ; Uric Acid ; metabolism

Enterovirus 71 (EV71) is a neurotropic pathogen that can induce hand, foot and mouth disease in children. There is an appreciable mortality rate after EV71 infections. The mechanism of action of EV71 replication is not known. Recent work has identified some of cell factors of the host that participate in the synthesis of the RNA and proteins of EV71 (e.g., hnRNP K, reticulon 3 (RTN 3)). In that work, researchers used a competitive assay to show that hnRNP K can interact with EV71 5' UTR, which is required for efficient synthesis of viral RNA. Using a yeast two-hybrid system, other researchers demonstrated that RTN 3 interacts with the N-terminal domain of EV71 2C, which is crucial for replication of viral RNA. Here, we discuss recent work focusing on the molecular mechanisms of hnRNP K and RTN 3 in the synthesis of the RNA and proteins of EV71.
Animals ; Carrier Proteins ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; physiology ; Enterovirus Infections ; genetics ; metabolism ; virology ; Heterogeneous-Nuclear Ribonucleoprotein K ; Host-Pathogen Interactions ; Humans ; Membrane Proteins ; genetics ; metabolism ; Nerve Tissue Proteins ; genetics ; metabolism ; Ribonucleoproteins ; genetics ; metabolism ; Viral Proteins ; genetics ; metabolism ; Virus Replication

OBJECTIVETo explore the effect of the cytoplasmic DNA sensor DAI on replication of hepatitis B virus (HBV) and its possible mechanism.

METHODSThe hepatocyte-derived cell line HepG2 was co-transfected with DAI siRNA and the HBV1.3 replicative plasmid PHY106, and the cells were divided into two experimental groups. Six hours later, total RNA was extracted from the first group of cells and expression of IFIT1 and IL-6 were detected by real-time RT-PCR. The second group of cells was incubated for 4 days, after which the cell supernatant was collected and the HBV surface antigen (HBsAg) and envelope antigen (HBeAg) were detected by ELISA. In addition, HBV core particles were extracted and applied to southern blot assay to detect the intracellular HBV replication intermediates (rcDNA, dlDNA and ssDNA). Next, the HepG2 cells were triple transfected with siRNA targeting the type I interferon pathway molecule TBK1 and DAI simultaneously and HBV1.3, after which HBV viral proteins were detected. Two-group comparisons were made using the independent sample t-test, and more-than-2-group comparisons were made using ANOVA.

RESULTSDAI gene expression was down-regulated in response to DAI siRNA transfection. Cells with down-regulated DAI showed inhibited HBV replication (in a dose-dependent manner), accompanied by reduced levels of HBsAg (0.0195+/-0.0050 vs.

CONTROL0.3150+/-0.0200, P less than 0.05, t = 14.77) and HBeAg (0.0140+/-0.0040 vs.

CONTROL0.01235+/-0.0135, P less than 0.05, t = 7.777). No effect of down-regulated DAI was observed for the expression of IFIT1 of IL-6. siRNA-mediated down-regulation of TBK1 and DAI simultaneously led to reduced expression of HBsAg and HBeAg.

CONCLUSIONDown-regulation of DAI gene expression inhibited HBV replication and HBV protein expression, but the underlying mechanism was not related to the type I interferon or NF-kB signaling pathway.

Carrier Proteins ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Down-Regulation ; Gene Expression Regulation ; Hep G2 Cells ; Hepatitis B Surface Antigens ; isolation & purification ; Hepatitis B e Antigens ; isolation & purification ; Hepatitis B virus ; physiology ; Humans ; Interleukin-6 ; metabolism ; NF-kappa B ; metabolism ; Plasmids ; RNA, Small Interfering ; genetics ; Signal Transduction ; Transfection ; Virus Replication

OBJECTIVETo study the effect of Zhusha Anshen pill, cinnabar, HgS, HgCl2 and MeHg on the gene expression of renal transporters in mice.

METHODHealthy male mice were given equivalent physiological saline, Zhusha Anshen pill (1.8 g · kg(-1), containing 0.17 g · kg(-1) of mercury), cinnabar (0.2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), high dose cinnabar (2 g · kg(-1), containing 1.7 g · kg(-1) of mercury), HgS (0.2 g · kg(-1), containing 0.17 g · kg(-1) of mercury), HgCl2 (0.032 g · kg(-1), containing 0. 024 g · kg(-1) of mercury), MeHg (0.026 g · kg(-1), containing 0.024 g · kg(-1) of mercury), once daily, for 30 d, measuring body mass gain. 30 days later, the mice were sacrificed. The mercury accumulation in kidneys was detected with atomic fluorescence spectrometer. Expressions of Oat1, Oat2, Oat3, Mrp2, Mrp4, Urat1 were detected with RT-PCR.

RESULTCompared with the normal control group, a significant accumulation of Hg in kidney in HgCl2 and MeHg groups was observed (P <0.05), but these changes were not found in other groups. Compared with normal control group, mRNA expressions of Oat1 and Oat2 were evidently lower in HgCl2 and MeHg groups, but mRNA expressions of Mrp2 were apparently higher in HgCl2 group (P <0.05), mRNA expression of Mrp4 was significant higher in HgCl2 and MeHg groups, and mRNA expression of Urat1 was apparently lower in MeHg group.

CONCLUSIONHgCl2 and MeHg groups show significant difference from the normal group in mercury accumulation in kidneys and gene expression of kidney transporters, but with no difference between other groups and the normal group. Compared with HgCl2 and MeHg, cinnabar and its compounds could cause lower renal toxicity to mice.

Animals ; Carrier Proteins ; genetics ; Drugs, Chinese Herbal ; toxicity ; Gene Expression ; drug effects ; Kidney ; drug effects ; metabolism ; Male ; Mercuric Chloride ; toxicity ; Mercury Compounds ; toxicity ; Methylmercury Compounds ; toxicity ; Mice ; Multidrug Resistance-Associated Proteins ; genetics ; Organic Anion Transport Protein 1 ; genetics ; Organic Anion Transporters, Sodium-Independent ; genetics

BACKGROUND: We investigated the rates of fecal transmission of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae (ESBL-E) and carbapenem-resistant Enterobacteriaceae (CRE) among patients admitted to intensive care units (ICUs). METHODS: From June to August 2012, rectal cultures were acquired from all patients at ICU admission. For patients not carrying ESBL-E or CRE at admission, follow-up cultures were performed to detect acquisition. A chromogenic assay was used to screen for ESBL-E and CRE. Bacterial species identification and antibiotic susceptibility tests were performed using the Vitek 2 system (bioMerieux, France). ESBL genotypes were determined by PCR, and clonal relatedness of the isolates was assessed by pulsed-field gel electrophoresis. RESULTS: Out of 347 ICU admissions, 98 patients were found to be carriers of ESBL-E (28.2%, 98/347). Follow-up cultures were acquired from 91 of the patients who tested negative for ESBL-E at admission; the acquisition rate in this group was 12.1% (11/91), although none was a nosocomial transmission. For CRE, the prevalence of fecal carriage was 0.3% (1/347), and the acquisition rate was 2.9% (4/140). None of the CRE isolates were carbapenemase-producers. CONCLUSIONS: The high prevalence of ESBL-E carriage on admission (28.2%), coupled with rare nosocomial transmission and the very low carriage rate of CRE (0.3%), challenge the routine use of active surveillance in non-epidemic settings. Nevertheless, passive surveillance measures, such as rapid and accurate screening of clinical specimens, will be critical for controlling the spread of CRE.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/*metabolism ; Carbapenems/*pharmacology ; Carrier State/epidemiology ; Cross Infection/epidemiology/*transmission ; DNA, Bacterial/analysis ; Drug Resistance, Bacterial/drug effects ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae/enzymology/genetics/*physiology ; Enterobacteriaceae Infections/epidemiology/*transmission ; Feces/*microbiology ; Genotype ; Humans ; Intensive Care Units ; Polymerase Chain Reaction ; Prevalence ; Republic of Korea/epidemiology ; beta-Lactamases/*metabolism