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Abstract EphrinB2 plays a critical role in tumor growth. In this study, we studied the antitumor activity of imperatorin derivative IMP-1 in renal cell carcinoma (RCC) by regulating EphrinB2 pathway.. Results showed that IMP-1 inhibited the proliferation of 786-O cells in a dose- and time-dependent manner. More importantly, knockdown and transfection of EphrinB2 altered the inhibitory effect of IMP-1 on the activity of 786-O cells. IMP-1 arrested 786-O cell cycle at G0/G1 phase by decreasing the expression of cyclin D1 and cyclin E. Moreover, IMP-1 regulated Bcl-2 family proteins' expression, thus inducing apoptosis of 786-O cells. IMP-1 down-regulated the expression of EphrinB2, Syntenin1 and PICK1. Then, IMP-1 decreased the phosphorylation of Erk1/2 and AKT. In all, IMP-1 could regulate the EphrinB2 pathway in order to inhibit 786-O cell growth by arresting the cell cycle at G0/G1 phase and inducing cell apoptosis. Thus, IMP-1 may present as a potential strategy for RCC treatment.
Subject(s)
Carcinoma, Renal Cell/pathology , Neoplasms/classification , G1 Phase/genetics , Cyclin D1/adverse effects , Cyclin E/adverse effectsABSTRACT
Objective To investigate the post-transcriptional regulation mechanism of insulin-like growth factor 2 (IGF2),which is an important tumor regulator,by SIRT6. Methods SIRT6 was overexpressed in 293T cells and IP was used to enrich SIRT6 and mass spectrometry(MS)was used to detect the protein that interacted with SIRT6. Western blot was used to validate the interacted protein and its acetylation/phosphorylation modification status. Results SIRT6 did not change the acetylation modification status of IMP1,but the phosphorylation status of IMP1 was elevated in the presence of SIRT6. Conclusions SIRT6 may regulate IGF2 though promoting the phosphoryla-tion of IMP1 in 293T cells.
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BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) with acquired metallo β-lactamase (MBL) resistance have been increasingly reported worldwide and associated with significant mortality and morbidity. Here, an outbreak of genetically related strains of Klebsiella pneumoniae producing the imipenemase (IMP)-1 MBL in a medical intensive care unit (MICU) in Korea is reported. METHODS: Since isolating carbapenem-resistant K. pneumoniae (CRKP) at the MICU of the hospital on August 10, 2011, surveillance cultures for CRE in 31 hospitalized patients were performed from August to September 2011. Carbapenem resistance was determined based on the disk diffusion method outlined in the Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was performed for genes coding for β-lactamase. Associations among isolates were assessed via pulsed-field gel electrophoresis (PFGE). In addition, a surveillance study of environmental cultures and health-care workers (HCWs) was conducted in the MICU during the same time frame. RESULTS: During the study period, non-duplicated CRKP specimens were discovered in four patients in the MICU, suggestive of an outbreak. On August 10, 2011, CRKP was isolated from the sputum of a 79-year-old male patient who was admitted to the MICU. A surveillance study to detect additional CRE carriers by rectal swab revealed an additional three CRKP isolates. PCR and sequencing of the four isolates identified the presence of the IMP-1 gene. In addition, PFGE showed that the four isolated strains were genetically related. CRE was not identified in specimens taken from the hands of HCWs or other environmental sources during surveillance following the outbreak. Transmission of the carbapenemase-producing Enterobacteriaceae strain was controlled by isolation of the patients and strict contact precautions. CONCLUSIONS: This study shows that rapid and systemic detection of CRE and strict infection controls are important steps in preventing nosocomial transmission.
Subject(s)
Aged , Humans , Male , Clinical Coding , Critical Care , Diffusion , Disease Outbreaks , Drug Resistance, Bacterial , Electrophoresis, Gel, Pulsed-Field , Enterobacteriaceae , Hand , Infection Control , Intensive Care Units , Klebsiella pneumoniae , Klebsiella , Korea , Methods , Mortality , Pneumonia , Polymerase Chain Reaction , SputumABSTRACT
BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) with acquired metallo β-lactamase (MBL) resistance have been increasingly reported worldwide and associated with significant mortality and morbidity. Here, an outbreak of genetically related strains of Klebsiella pneumoniae producing the imipenemase (IMP)-1 MBL in a medical intensive care unit (MICU) in Korea is reported. METHODS: Since isolating carbapenem-resistant K. pneumoniae (CRKP) at the MICU of the hospital on August 10, 2011, surveillance cultures for CRE in 31 hospitalized patients were performed from August to September 2011. Carbapenem resistance was determined based on the disk diffusion method outlined in the Clinical and Laboratory Standards Institute guidelines. Polymerase chain reaction (PCR) was performed for genes coding for β-lactamase. Associations among isolates were assessed via pulsed-field gel electrophoresis (PFGE). In addition, a surveillance study of environmental cultures and health-care workers (HCWs) was conducted in the MICU during the same time frame. RESULTS: During the study period, non-duplicated CRKP specimens were discovered in four patients in the MICU, suggestive of an outbreak. On August 10, 2011, CRKP was isolated from the sputum of a 79-year-old male patient who was admitted to the MICU. A surveillance study to detect additional CRE carriers by rectal swab revealed an additional three CRKP isolates. PCR and sequencing of the four isolates identified the presence of the IMP-1 gene. In addition, PFGE showed that the four isolated strains were genetically related. CRE was not identified in specimens taken from the hands of HCWs or other environmental sources during surveillance following the outbreak. Transmission of the carbapenemase-producing Enterobacteriaceae strain was controlled by isolation of the patients and strict contact precautions. CONCLUSIONS: This study shows that rapid and systemic detection of CRE and strict infection controls are important steps in preventing nosocomial transmission.
Subject(s)
Aged , Humans , Male , Clinical Coding , Critical Care , Diffusion , Disease Outbreaks , Drug Resistance, Bacterial , Electrophoresis, Gel, Pulsed-Field , Enterobacteriaceae , Hand , Infection Control , Intensive Care Units , Klebsiella pneumoniae , Klebsiella , Korea , Methods , Mortality , Pneumonia , Polymerase Chain Reaction , SputumABSTRACT
Objective To investigate serum anti-insulin-like growth factor-Ⅱ mRNA binding protein1(IMP1) autoantibody IgG expression in hepatocellular carcinoma(HCC) patient and its relationship with clinicopathological parameters.Methods ELISA method was used to detect serum anti-IMP1 autoantibody IgG in 120 cases of HCC,then the detection results were compared those in 115 healthy individuals(control group).The relationship between serum anti-IMP1 autoantibody IgG level with clinicopathological parameters in HCC patients was analyzed.Results The serum anti-IMP1 autoantibody IgG levels in the HCC group and control group were(1.512±0.685),(1.080±0.301)μg/mL respectively,the HCC group was significantly higher than the control group with statistical difference(P0.05).Conclusion Serum anti-IMP1 autoantibody IgG expression increased is increased in HCC patient.The detection of serum anti-IgG autoantibody IMP1 level in HCC is conducive to judge the malignancy degree of HCC.
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Objective To investigate the phenotype and genotype in a carbapenem resistant L.adecarboxylata strain.Methods Microbial identification and drug susceptibility test was done with VITEK II Compact.Carbapenemases genes were detec-ted by PCR methods.Results The strain of carbapenem resistant L.adecarboxylata produced IMP-1 carbapenemase.Con-clusion L.adecarboxylata was found only rarely in the clinical isolates.This was the first Isolate of L.adecarboxylata pro-ducing IMP-1 metallo-beta-lactamase.
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Objective To subclone express and identify the immune mapped protein 1 IMP1 which encodes a surface an?tigen of Toxoplasma gondii. Methods The cDNA of T. gondii RH strain was synthesized by reverse transcription PCR the IMP1 open reading frame ORF was amplified by PCR using the T. gondii RH strain cDNA as template the PCR products were identified by TA?cloning and sequencing then the IMP1 ORF was subcloned into the NdeⅠand Xho I sites of the vector pET28b and the positive recombinant pET28b?IMP1 was identified by double?digesting and sequencing. The protein of 6 × His tagged IMP1 was inducibly expressed in E. coli strain BL21 DE3 with isopropylβ?D?1?thiogalactopyranoside IPTG and the induction time concentration of IPTG and temperature gradients to optimize protein expression conditions were determined. After the cells carried IMP1 were induced by the optimized conditions and harvested the resulting bacteria were suspended in resuspension buffer and lysed by sonication and the supernatants were loaded onto the Ni2+Chelating Sepharose Fast Flow col?umn for affinity chromatography of the N?terminal 6 × His tagged IMP1 protein. Finally the fusion IMP1 proteins were identified by Western blotting. Results The ORF sequence of IMP1 was successfully subcloned from the cDNA of Toxoplasma Gondii RH strain and the amplified product was sequenced and identified based on which the IMP1 ORF gene was inserted into the prokaryotic expression vector pET28b and the recombinant pET28b?IMP1 was constructed successfully. The double?digesting and sequencing results indicated the validity of the recombinant vector. And the optimized conditions for the expression of IMP 1 was determined namely 0.3 mmol/L IPTG induction for 9 h at 20℃. Furthermore IMP1 protein was expressed solubly and che?lated on Ni2+sepharose beads with high affinity thus this protein could be purified efficiently by affinity chromatography. The pure fusion protein was confirmed with fine immunocompetence by SDS?PAGE and Western blotting. Conclusions IMP1 pro? tein can be high efficiently expressed by the E. coli prokaryotic expression systems the protein of IMP1 is soluble and has stable characters. The study may lay a useful foundation for the following works including in vivo expression of IMP1 crystal structure study of IMP1 and anti?toxoplasmosis subunit vaccine development.
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BACKGROUND: Metallo-beta-lactamases (MBLs) have been reported in gram negative bacilli and are becoming increasingly important clinically because the enzymes hydrolyse almost all beta-lactams, including carbapenems. Thus, the present study was conducted to determine the prevalence of MBL types in imipenem-nonsusceptible Pseudomonas aeruginosa and Acinetobacter baumannii isolated from a tertiary teaching hospital. METHODS: Imipenem-nonsusceptible strains, 128 P. aeruginosa and 93 A. baumannii, were collected from clinical specimens. Identification and susceptibility tests were determined by Vitek GNI and GNS cards. MBL production was determined by modified Hodge test and imipenem-EDTA synergy test. Multiplex PCR amplification of MBL genes including blaIMP-1, blaVIM-1 and blaVIM-2 were performed. RESULTS: Thirty-one P. aeruginosa (24.2%) isolates and 3 A. baumannii (3.2%) were found to be MBL producers. In P. aeruginosa, 20 (15.6%) and 11 (8.6%) isolates were positive for blaIMP-1 and blaVIM-2, respectively whereas 1 (1.0%) and 2 (2.2%) isolates in A. baumannii, respectively. CONCLUSION: IMP-1 is more prevalent MBL type than VIM-2 among imipenem-nonsusceptible P. aeruginosa unlike in other studies. Larger numbers of isolates and sequential studies are strongly recommended for the useful evaluation and monitoring of MBL production in the hospital setting to infection-control.
Subject(s)
Acinetobacter , Acinetobacter baumannii , beta-Lactams , Carbapenems , Drug Resistance, Multiple , Multiplex Polymerase Chain Reaction , Prevalence , Pseudomonas , Pseudomonas aeruginosaABSTRACT
PURPOSE: Two Korean nationwide studies showed that metallo-beta-lactamases (MBLs)-producing-Pseudomonas spp. are not rare. The aim of this study was to assess the trends of MBL-producing isolates among imipenem-resistant isolates of Pseudomonas spp. MATERIALS AND METHODS: Imipenem-resistant clinical isolates were collected from 23 hospitals and one commercial laboratory participating in the KONSAR program in 2005. Polymerase chain reaction (PCR) was used to detect MBL genes. RESULTS: Alleles of MBL genes were detected in 10.8% of 415 Pseudomonas aeruginosa and 66.7% of 12 P. putida isolates from 18 of 24 hospitals/laboratory. Among the 14 IMP-1-like and 39 VIM-2-like MBLs, emergence of IMP-6 was detected for the first time. CONCLUSION: Prevalence of MBL-producing P. aeruginosa has not significantly increased, but IMP-6 emerged in P. aeruginosa.
Subject(s)
Humans , Anti-Bacterial Agents/pharmacology , Electrophoresis, Gel, Pulsed-Field , Imipenem/pharmacology , Korea , Polymerase Chain Reaction , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , beta-Lactamases/geneticsABSTRACT
BACKGROUND: Spread of imipenem-resistant Pseudomonas aeruginosa isolates is an important clinical threat. The aim of this study is to survey the prevalence of carbapenem-resistant P.aeruginosa isolates in a university hospital, Busan, Korea, and to determine the mechanisms of the resistance. METHODS: P.aeruginosa isolates from the patients in Kosin University Gospel Hospital were collected during the period of June through September, 2004. Antimicrobial susceptibilities were tested by the disk diffusion method, and production of carbapenemase and metallo-beta-lactamase was determined by the modified Hodge and EDTA-disk synergy tests, respectively. MICs were determined by the agar dilution method, and pIs of beta-lactamases were determined by the isoelectric focusing. Genotypes of carbapenemases were determined by direct sequencing of amplified products. RESULTS: A total of 77 clinical isolates of P.aeruginosa were collected. Twenty-two (55.0%) and 15 (37.5%) isolates showed positive results in the modified Hodge and EDTA-disk synergy tests, re-spectively. Searches for bla OXA-23 and bla IMP-1 genes showed positive results in 15 and 12 isolates, respectively. MIC ranges of imipenem and meropenem to OXA-23-producing isolates were 8-16 microgram/mL and 2-32 microgram/mL, respectively, and those to IMP-1-producing isolates were 2-> or =256 microgram/mL and 2-128 microgram/mL, respectively. CONCLUSION: Production of OXA-23 or IMP-1 is the most prevalent mechanism of imipenem-resistance in P.aeruginosa isolates in a university hospital, Busan, Korea. Periodical surveys are necessary to monitor the spreading of imipenem-resistant isolates and emerging new mechanisms of imipenem-resistance.
Subject(s)
Humans , Agar , beta-Lactamases , Diffusion , Genotype , Imipenem , Isoelectric Focusing , Korea , Prevalence , Pseudomonas aeruginosa , PseudomonasABSTRACT
BACKGROUND: The purposes of this study were to investigate the prevalence of imipenem-resistant clinical Acinetobacter baumannii isolates and to determine the mechanism of the resistance. METHODS: During the period of June to September 2004, susceptibility to imipenem of A. baumannii isolates from a hospital in Busan, Korea were investigated. The isolates were screened for the production of carbapenemase and metallo-beta-lactamase by Modified-Hodge and EDTA-disk synergy tests, respectively; minimum inhibitory concentrations (MICs) were determined by agar dilution method. Genes coding for GES, IMP, VIM, SMP-1, GIM-1 and OXA type beta-lactamases were searched by PCR amplification, and the PCR products were subjected to direct sequencing. Isoelectric points of beta-lactamases were estimated by isoelectric focusing and the epidemiological relationships of isolates were investigated by enterobacterial repetitive intergenic consensus (ERIC) PCR. RESULTS: Fifty eight strains of A. baumannii were isolated from clinical specimens during the surveillance period, and 14 isolates (24.1%) were resistant to imipenem. Of the 14 isolates, 9 were tested positive in Modified-Hodge test and 2 were also positive in EDTA-disk synergy test. Genes encoding OXA-23 and IMP-1 were detected in 7 and 2 isolates, respectively. In IEF studies, OXA-23 and IMP-1 enzymes had corresponding pIs at 6.7 and 9.0, respectively. Seven OXA-23-producing and 2 IMP-1-producing isolates showed the same ERIC PCR patterns. CONCLUSION: It is concluded that 7 and 2 A. baumannii isolates from the patients in a hospital in Busan acquired resistance to imipenem by producing OXA-23 and IMP-1 beta-lactamases, respectively. The isolates producing these beta-lactamases might be originated from a common source.
Subject(s)
Humans , Acinetobacter baumannii , Acinetobacter , Agar , beta-Lactamases , Clinical Coding , Consensus , Imipenem , Isoelectric Focusing , Isoelectric Point , Korea , Microbial Sensitivity Tests , Molecular Epidemiology , Polymerase Chain Reaction , PrevalenceABSTRACT
BACKGROUND: The therapeutic difficulty due to wide-spread emergence of multiply resistant strains is a major problem in Pseudomonas aeruginosa infection. Carbapenem-resistant P. aeruginosa strains are being isolated with increasing frequency. Clinical isolates of P. aeruginosa with transferable imipen-em resistance due to production of metallo-beta-lactamase (MBL) have been reported. This study was performed to determine the usefulness of the imipenem-EDTA disk test to detect MBL, to examine the prevalence of MBL in a tertiary care hospital in Korea. METHODS: One hundred sixteen P. aeruginosa isolates with reduced susceptibilities to imipenem were collected during the period of 2000-2003 in the Samsung Medical Center. Imipenem-resistant P. aeruginosa isolates were examined for MBL production by imipenem-EDTA disk tests. To detect of blaIMP-1 , blaVIM-1, and blaVIM-2 genes, polymerase chain reactions (PCR) were performed and the positive isolates were confirmed by sequencing. RESULTS: Among 116 clinical isolates of P. aeruginosa, 20 isolates (17.2%) were positive for the imipenem-EDTA disk tests. Nineteen isolates (16.4%) carried VIM-2. Accoroding to PCR results, the sensitivity, specificity, and test efficiency of the imipenem-EDTA disk tests were 89%, 97%, and 96%, respectively. CONCLUSIONS: The imipenem-EDTA disk test is sensitive and specific for detecting VIM producer. VIM-2 may be an important MBL in P. aeruginosa in tertiary care hospitals the Korea. The spread of MBL genes could compromise the future usefulness of carbapenem for the treatment of gram-neg-ative bacilli infections.
Subject(s)
Imipenem , Korea , Mass Screening , Polymerase Chain Reaction , Prevalence , Pseudomonas aeruginosa , Sensitivity and Specificity , Tertiary HealthcareABSTRACT
Objective:To establish the method for the expression,purification and characterization of metallo-?-lactamases(IMP-1) and to explore the influence of experimental conditions on its separation and purification.Methods:pET9a/d-IMP-1 plasmid was transformed into E.coli competent BL21(DE3) cell and inoculated in LB medium.The supernatant were applied to SP Sepharose Fast Flow column,following by Sephadex G-75 column,and the purified enzymes were identified by SDS-PAGE and MALDI-TOF-MS and their activity was determined using Nitrocefin as substrate.Results:The purified enzyme showed high catalytic activity in the hydrolysis of most antibiotics.Its Michaelis constant K_m9.28?mol/L,and molecular weight Mr equalled 25111.9(determined by MALDI-TOF-MS).Conclusion:The method was established for the expression,purification and characterization of metallo-?-lactamases(IMP-1),which can be applicable to other metallo-?-lactamases.