To explore the mechanism for the role of autophagy in endometriosis, and to provide a theoretical basis for prevention and treatment of endometriosis.
 Methods: The endometrial CRL-7566 cells were treated with ATG5 siRNA, autophagic activator rapamycin and autophagic inhibitor 3-MA, respectively. The cell proliferation and invasion were detected by clonal formation, cell growth curve and MTT assay. The clinical specimens of endometriosis were collected from 20 cases. The expression of autophagy marker LC3II and autophagy substrate protein P62 were detected.
 Results: Rapamycin inhibited the proliferation and clonal formation of CRL-7566 cells, while autophagy inhibitor 3-MA and ATG5 siRNA showed opposite effect. Moreover, rapamycin inhibited filopodia growth in endometriosis, whereas overexpression of filopodia-relevant protein fascin-1 inhibited the decrease in invasiveness caused by rapamycin. In clinical samples, we also found a significant decrease of LC3II while an increase in P62 compared with the control group.
 Conclusion: Autophagy inhibition may contribute to an increase in endometrial cell proliferation and invasiveness. Autophagy activation could be a potential strategy for endometriosis therapy.
Autophagy ; drug effects ; genetics ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; drug effects ; Endometriosis ; physiopathology ; Endometrium ; cytology ; Female ; Gene Expression Regulation ; Humans ; Microfilament Proteins ; genetics ; metabolism ; Microtubule-Associated Proteins ; genetics ; RNA-Binding Proteins ; genetics ; Sirolimus ; pharmacology

Autophagy is an evolutionarily conserved cellular process which degrades intracellular contents. The Atg17-Atg31-Atg29 complex plays a key role in autophagy induction by various stimuli. In yeast, autophagy occurs with autophagosome formation at a special site near the vacuole named the pre-autophagosomal structure (PAS). The Atg17-Atg31-Atg29 complex forms a scaffold for PAS organization, and recruits other autophagy-related (Atg) proteins to the PAS. Here, we show that Atg31 is a phosphorylated protein. The phosphorylation sites on Atg31 were identified by mass spectrometry. Analysis of mutants in which the phosphorylated amino acids were replaced by alanine, either individually or in various combinations, identified S174 as the functional phosphorylation site. An S174A mutant showed a similar degree of autophagy impairment as an Atg31 deletion mutant. S174 phosphorylation is required for autophagy induced by various autophagy stimuli such as nitrogen starvation and rapamycin treatment. Mass spectrometry analysis showed that S174 is phosphorylated constitutively, and expression of a phosphorylation-mimic mutant (S174D) in the Atg31 deletion strain restores autophagy. In the S174A mutant, Atg9-positive vesicles accumulate at the PAS. Thus, S174 phosphorylation is required for formation of autophagosomes, possibly by facilitating the recycling of Atg9 from the PAS. Our data demonstrate the role of phosphorylation of Atg31 in autophagy.
Alanine ; chemistry ; metabolism ; Amino Acid Motifs ; Aspartic Acid ; chemistry ; metabolism ; Autophagy ; genetics ; Autophagy-Related Proteins ; Carrier Proteins ; chemistry ; metabolism ; Gene Expression Regulation, Fungal ; Membrane Proteins ; chemistry ; metabolism ; Models, Molecular ; Molecular Sequence Data ; Nitrogen ; deficiency ; Phagosomes ; chemistry ; drug effects ; metabolism ; Phosphorylation ; Protein Transport ; Saccharomyces cerevisiae ; drug effects ; genetics ; metabolism ; Saccharomyces cerevisiae Proteins ; chemistry ; genetics ; metabolism ; Serine ; chemistry ; metabolism ; Signal Transduction ; Sirolimus ; pharmacology

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

OBJECTIVETo screen the differentially expressed genes in human renal clear-cell carcinoma (RCC) cells using suppression subtractive hybridization (SSH), and to explore their biological function and underlying mechanism in RCC cells.

METHODSTotal RNAs were extracted from human renal clear-cell carcinoma cell line RLC-310 and human normal renal cell line HK-2 cells, and SSH technology was used to construct a RCC cell library of differential expression genes and to screen the most differentially expressed genes. RNA interference vector was constructed to silence the expression of the differentially expressed gene SIPL1 in human renal cell lines RLC-310 and GRC-1. Proliferation index was estimated by cell counting, MTT and tumor xenograft assay. Cell cycle analysis was performed using fluorescence activated cell sorting. Drug resistance potential to adriamycin was assessed by MTT.

RESULTSA subtractive cDNA library of highly expressed genes in the RCC cells was constructed and 12 differentially expressed genes were screened from the subtractive library, in which SIPL1 was the most differently expressed gene in the RCC cell line. SIPL1 overexpression in the RCC cells and clinical samples was confirmed by RT-PCR and Western blot analyses. The shRNA expression plasmid targeting to SIPL1 gene was constructed and transfected into RLC-310 and GRC-1 cells, resulting in downregulation of SIPL1. SIPL1 knockdown inhibited the cell proliferation (P < 0.05) and tumorgenesis. The tumor weights formed by RLC-310 cells transfected with SIPL1 shRNA was (0.22 ± 0.07)g and that of negative control vector was (0.85 ± 0.06)g. The tumor weight formed by GRC-1 cells was (0.32 ± 0.07)g and that of control vectors was (1.21 ± 0.11)g (P < 0.05). SIPL1 shRNA-transfected RLC-310 cells showed that more cells were arrested at G0/G1 phase [(71.13 ± 4.58)%] than that in the negative control RLC-310 cells [(53.27 ± 3.34)%, P < 0.05]. The proportion of G0/G1 phase in the SIPL1 shRNA transfected GRC-1 cells was (73.83 ± 3.97)%, significantly higher than that of (59.33 ± 3.03)% in the negative control GRC-1 cells (P < 0.05), and enhanced their sensitivity to adriamycin (P < 0.05). Silence of SIPL1 caused inactivation of AKT signaling and up-regulated expression of P27(Kip1) and P21(Cip1) proteins.

CONCLUSIONSA differentially expressed gene SIPL1 in the renal clear-cell carcinoma is successfully screened using SSH technology. SIPL1 functions as an oncogene in RCC, and may become a novel molecular target for RCC diagnosis and therapy.

Adenocarcinoma ; metabolism ; pathology ; Adult ; Aged ; Animals ; Antibiotics, Antineoplastic ; pharmacology ; Carcinoma, Renal Cell ; metabolism ; pathology ; Carrier Proteins ; genetics ; metabolism ; Cell Cycle ; Cell Line ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p27 ; metabolism ; Doxorubicin ; pharmacology ; Drug Resistance, Neoplasm ; Female ; Humans ; Kidney ; cytology ; Kidney Neoplasms ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Middle Aged ; Neoplasm Transplantation ; Nucleic Acid Hybridization ; Proto-Oncogene Proteins c-akt ; metabolism ; RNA Interference ; RNA, Messenger ; metabolism ; RNA, Small Interfering ; genetics ; Transfection ; Tumor Burden

OBJECTIVETo study the effect of silencing pyruvate kinase M2 (PKM2) on gambogic acid (GA)-induced apoptosis of human prostate cancer PC3 cells.

METHODSThree specific PKM2 siRNAs and one negative control siRNA (si-NC) were transfected into PC3 cells. The silencing effect of PKM2 siRNAs was determined by real-time fluorescence quantitative PCR (qRT-PCR) and Western blot, and the effects of PKM2 siRNA on the vitality and apoptosis of GA-stimulated PC3 cells detected by MTT and AO/EB double staining, respectively. The mRNA and protein levels of c-myc and cyclin D1 were analyzed by qRT-PCR and Western blot, respectively.

RESULTSAll the 3 PKM2 siRNAs effectively reduced the mRNA and protein expressions of PKM2, and PKM2 siRNA-1 exhibited the strongest silencing effect. At 24 h after transfection, the expression levels of PKM2 mRNA and protein were reduced by 70% and 85%, respectively (P < 0.05). Twenty-four hours of treatment with GA (0.5 micromol/L) following transfection with PKM2 siRNA-1 inhibited the vitality of the PC3 cells by 68%, increased their apoptosis, and significantly down-regulated the mRNA and protein levels of c-myc (50% and 35%) and cyclin D1 (60% and 20%) (P < 0.05).

CONCLUSIONInhibition of PKM2 sensitized PC3 cells to GA-induced apoptosis, suggesting that PKM2 may be a potential therapeutic target for sensitizing human prostate cancer to GA.

Apoptosis ; drug effects ; Carrier Proteins ; genetics ; metabolism ; Cell Line, Tumor ; Humans ; Male ; Membrane Proteins ; genetics ; metabolism ; Prostatic Neoplasms ; genetics ; metabolism ; pathology ; RNA Interference ; RNA, Small Interfering ; Thyroid Hormones ; genetics ; metabolism ; Xanthones ; pharmacology

Long-term synaptic plasticity requires addition of new proteins at the synaptic site. The local protein synthesis at subsynaptic sites confers advantageous mechanisms that would regulate the protein composition in local domains on a moment-by-moment basis. However, our information on the identities of 'dendritic' mRNAs is very limited. In this study we investigated the expression of the protein and mRNA for eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4EBP1) in cultured rat hippocampal neurons. Immunocytochemistry (ICC) showed that 4EBP1 protein is highly localized to the nucleus. In dendrites most 4EBP1 punctae were not colocalized with those of eIF4E. In situ hybridization (ISH) and Fluorescence ISH (FISH) revealed that 4EBP1 mRNA was present in dendrites. The FISH signals formed clusters along dendrites that colocalized with ICC signals for Staufen, a marker for RNA granules. The neuronal activation by KCl (60 mM, 10 min) significantly increased the density of 4EBP1 FISH signals in the nucleus after 2 hr, and both in the nucleus and dendrites after 6 hr. Our results indicate that 4EBP1 and its mRNA are present in dendrites, and the mRNA is upregulated and transported to dendritic domains in RNA granules upon neuronal activation.
Animals ; Carrier Proteins/genetics/*metabolism ; Cell Nucleus/metabolism ; Cells, Cultured ; Dendrites/*metabolism ; Hippocampus/cytology/drug effects/*metabolism ; Immunohistochemistry ; In Situ Hybridization, Fluorescence ; Phosphoproteins/genetics/*metabolism ; Potassium Chloride/pharmacology ; RNA, Messenger/*metabolism ; RNA-Binding Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Up-Regulation/drug effects

Higher levels of body fat are associated with an increased risk for development numerous adverse health conditions. FTY720 is an immune modulator and a synthetic analogue of sphingosine 1-phosphate (S1P), activated S1P receptors and is effective in experimental models of transplantation and autoimmunity. Whereas immune modulation by FTY720 has been extensively studied, other actions of FTY720 are not well understood. Here we describe a novel role of FTY720 in the prevention of obesity, involving the regulation of adipogenesis and lipolysis in vivo and in vitro. Male C57B/6J mice were fed a standard diet or a high fat diet (HFD) without or with FTY720 (0.04 mg/kg, twice a week) for 6 weeks. The HFD induced an accumulation of large adipocytes, down-regulation of phosphorylated AMP-activated protein kinase alpha (p-AMPKalpha) and Akt (p-Akt); down-regulation of hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL) and perilipin mRNA as well as up-regulation of phosphorylated HSL (p-HSL, Ser563) and glycogen synthase kinase 3 alpha/beta (p-GSK3alpha/beta). All these effects were blunted by FTY720 treatment, which inhibited adipogenesis and promoted lipolysis. Also, FTY720 significantly decreased lipid accumulation in maturing preadipocytes. FTY720 down-regulated the transcriptional levels of the PPARgamma, C/EBPalpha and adiponectin, which are markers of adipogenic differentiation. FTY720 significantly increased the release of glycerol and the expression of the HSL, ATGL and perilipin, which are regulators of lipolysis. These results show that FTY720 prevented obesity by modulating adipogenesis and lipolysis, and suggest that FTY720 is used for the treatment of obesity.
3T3-L1 Cells ; AMP-Activated Protein Kinases/metabolism ; Adipocytes/*drug effects/physiology ; Adipogenesis/drug effects ; Animals ; Anti-Obesity Agents/*pharmacology/therapeutic use ; Antigens, Differentiation/genetics/metabolism ; Carrier Proteins/genetics/metabolism ; Cell Size ; Diet, High-Fat/adverse effects ; Disease Models, Animal ; Enzyme Activation ; Gene Expression Regulation, Enzymologic/drug effects ; Glycogen Synthase Kinase 3/genetics/metabolism ; Lipase/genetics/metabolism ; Lipolysis/drug effects ; Male ; Mice ; Mice, Inbred C57BL ; Obesity/etiology/metabolism/*prevention & control ; Phosphoproteins/genetics/metabolism ; Phosphorylation ; Propylene Glycols/*pharmacology/therapeutic use ; Protein Processing, Post-Translational ; Proto-Oncogene Proteins c-akt/metabolism ; Sphingosine/*analogs & derivatives/pharmacology/therapeutic use ; Sterol Esterase/metabolism

OBJECTIVETo explore the molecular mechanism of puerarin (Pue) in improving insulin resistance through observing its effect on the insulin resistance of 3T3-Li lipocyte induced by free fatty acid (FFA).

METHODS3T3-L1 preadipocyte was induced by a culture solution containing insulin, isobutyo-menthyl-xanthine, and dexamethasone to mature lipocyte, and it was divided into six groups: the control group (normal cells), the model group (untreated model cells), and the four drug treatment group exposed to dimethyl biguanide (Met group), highdose puerarin (PueH group), low-dose puerarin (PueL group), and propylene glycol (PG group), respectively. Mature lipocytes in various groups, except those in the normal group, were established into insulin resistance model by FFA induction and treated respectively with corresponding drugs. Peroxisome proliferator-activated receptor-γ (PPAR-γ) mRNA expressions at the fourth, sixth, and eighth day were observed using reverse transcription polymerase chain reaction (RT-PCR); glucose transportation in various groups were observed by 2-deoxy-[(3)H]-D-glucose intake method; mRNA expression of Cb1 binding protein (CAP) was determined by RT-PCR; and glucose transporter-4 (Glut-4) transposition was detected by immune-fluorescence method.

RESULTSPPAR-γmRNA expression increased gradually, and it showed lower levels at the fourth, sixth, and eighth day in all treatment groups than that in the model group. Glucose transportation determination showed that the transportation in the model group was 2.23±0.63, significantly lower than that in the normal group 5.05 ± 0.66 (P<0.01); as compared with the model group, they were significantly higher in the PueH and the PueL groups. In addition, the CAP mRNA expression and membranous distribution of Glut-4 were higher in the two Pue treated groups than those in the model group, respectively.

CONCLUSIONPue could markedly improve the insulin resistance of 3T3-L1 lipocyte, which is realized possibly by way of inactivating CAP path, promoting Glut-4 transposition to cell membrane to increase the transportation of glucose.

3T3-L1 Cells ; Adipocytes ; drug effects ; metabolism ; Animals ; Biological Transport ; drug effects ; Carrier Proteins ; metabolism ; Gene Expression Regulation ; drug effects ; Glucose ; metabolism ; Glucose Transporter Type 4 ; metabolism ; Insulin Resistance ; Isoflavones ; pharmacology ; Mice ; PPAR gamma ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; drug effects

The effects of mangiferin on uric acid excretion, kidney function and related renal transporters were investigated in hyperuricemic mice induced by potassium oxonate. Mice were divided into normal control group, and 5 hyperuricemic groups with model control, 50, 100, and 200 mg x kg(-1) mangiferin, and 5 mg x kg(-1) allopurinol. Mice were administered by gavage once daily with 250 mg x kg(-1) potassium oxonate for seven consecutive days to create the model. And 3 doses of mangiferin were orally initiated on the day 1 h after potassium oxonate was given, separately. Serum uric acid, creatinine and urea nitrogon levels, as well as urinary uric acid creatinine levels were measured. Mouse uromodulin (mUMOD) levels in serum, urine and kidney were determined by ELISA method. The mRNA and protein levels of related renal transporters were assayed by RT-PCR and Western blotting methods, respectively. Compared to model group, mangiferin significantly reduced serum uric acid, creatinine and urea nitrogon levels, increased 24 h uric acid and creatinine excretion, and fractional excretion of uric acid in hyperuricemic mice, exhibiting uric acid excretion enhancement and kidney function improvement. Mangiferin was found to down-regulate mRNA and protein levels of urate transporter 1 (mURAT1) and glucose transporter 9 (mGLUT9), as well as up-regulate organic anion transporter 1 (mOAT1) in the kidney of hyperuricemic mice. These findings suggested that mangiferin might enhance uric acid excretion and in turn reduce serum uric acid level through the decrease of uric acid reabsorption and the increase of uric acid secretion in hyperuricemic mice. Moreover, mangiferin remarkably up-regulated expression levels of renal organic cation and carnitine transporters (mOCT1, mOCT2, mOCTN1 and mOCTN2), increased urine mUMOD levels, as well as decreased serum and kidney mUMOD levels in hyperuricemic mice, which might be involved in mangiferin-mediated renal protective action.
Animals ; Blood Urea Nitrogen ; Carrier Proteins ; genetics ; metabolism ; Creatinine ; blood ; Glucose Transport Proteins, Facilitative ; genetics ; metabolism ; Hyperuricemia ; blood ; chemically induced ; physiopathology ; urine ; Kidney ; metabolism ; physiopathology ; Male ; Membrane Proteins ; genetics ; metabolism ; Mice ; Octamer Transcription Factor-1 ; genetics ; metabolism ; Organic Anion Transport Protein 1 ; genetics ; metabolism ; Organic Anion Transporters ; genetics ; metabolism ; Organic Cation Transport Proteins ; genetics ; metabolism ; Organic Cation Transporter 2 ; Oxonic Acid ; Protective Agents ; pharmacology ; RNA, Messenger ; metabolism ; Random Allocation ; Solute Carrier Family 22 Member 5 ; Uric Acid ; blood ; urine ; Uromodulin ; blood ; urine ; Xanthones ; pharmacology

Cyanovirin-N (CVN) is an 11 kDa anti-HIV protein originally isolated from extracts of a cyanobacterium, Nostoc ellipsosporum. The protein binds with high affinity to the viral envelope glycoprotein gp120 and irreversibly inactivates diverse HIV strains. A fusion gene consisting of cvn, sumo and 6xHis tag was synthesized by PCR according to the codon bias of Escherichia coli. The fusion protein is expressed in the cytoplasm of E. coli in a soluble form and up to 28% of the total protein. The recombinant CVN was purified to homogeneity by 2 rounds of Ni-NTA affinity chromatography and one round of SUMO protease cleavage. Bioactivity assay demonstrated that SUMO-CVN and CVN bound to gp120 with nanomolar concentration. In addition, CVN showed potent anti-HSV-1 and anti-HIV-1 activities in in vitro cellular assays. Therefore, the 6xHis SUMO fusion expression and purification system provides a better approach for large scale production of CVN for further microbicide development.
Antiviral Agents ; isolation & purification ; metabolism ; pharmacology ; Bacterial Proteins ; biosynthesis ; genetics ; pharmacology ; Carrier Proteins ; biosynthesis ; genetics ; pharmacology ; Escherichia coli ; genetics ; metabolism ; HIV-1 ; drug effects ; Herpesvirus 1, Human ; drug effects ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification ; pharmacology