No abstract available.
Isoelectric Focusing* ; Polymorphism, Genetic*

No abstract available.
HLA-A2 Antigen* ; Isoelectric Focusing*

OBJECTIVETo investigate the expression of androgen receptor (AR) isoforms in normal human prostate.

METHODSFourteen normal prostatic specimens from donors, aged 25 on average (21-28 yr), were analyzed by high resolution isoelectric focusing (IEF). The expression of AR isoforms was demonstrated in all 14 normal human prostatic tissues.

RESULTSFour types of AR isoforms were detected with isoelectric point value at 6.5, 6.0, 5.8 and 5.3 in 14 prostatic specimens. Binding of 3H-dihydrotestosterone (DHT) to these four AR isoforms was inhibited by the addition of 100-fold excess of DHT and testosterone. No effect of progesterone, estradiol and diethylstilbestrol on tritiated hormone binding was observed.

CONCLUSIONSThere are four AR isoforms in normal human prostate. The expression of AR isoforms is different from one another.

Adult ; Humans ; Isoelectric Focusing ; Isoelectric Point ; Male ; Prostate ; metabolism ; Protein Isoforms ; biosynthesis ; Receptors, Androgen ; biosynthesis

BACKGROUND AND OBJECTIVE: Buckwheat is considered one of the most important food allergens in Korea. Although a very small amount is ingested or inhaled, it can cause serious allergic reactions. However, the major allergens of buckwheat still remain to be elucidated. The aim of our study was to identify and characterize the major allergen of buckwheat seed. MATERIAL AND METHOD: Dialysis membrane with a cut-off MW 1kD was used for the preparation of crude buckwheat seed allergen extract. SDS-PAGE under reducing conditions and IgE immunoblotting were performed using sera from 15 buckwheat sensitive subjects. Isoelectric focusing and lectin blotting assay were done. RESULT: Western blot analysis showed more than 15 IgE-reactive buckwheat proteins. Among them, a 24kD protein was shown to be the most frequently bound to sera from allergic subjects (54%). Isoelectric point of 24kD protein was around 5.9. In lectin blotting assay, 24kD protein did not bind to Con A nor five other lectins. CONCLUSION: A 24kD protein was the most frequently recognized allergenic component in buckwheat seed. Isoelectric point was around 5.9. Glycosylation was not detected in 24kD of buckwheat protein.
Allergens ; Blotting, Western ; Dialysis ; Electrophoresis, Polyacrylamide Gel ; Fagopyrum* ; Glycosylation ; Hypersensitivity ; Immunoblotting ; Immunoglobulin E ; Isoelectric Focusing ; Isoelectric Point ; Korea ; Lectins ; Membranes

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.
Agar ; beta-Lactamases ; Busan ; Diffusion ; Genotype ; Humans ; Imipenem ; Isoelectric Focusing ; Korea ; Prevalence* ; Pseudomonas aeruginosa* ; Pseudomonas*

BACKGROUND: Increase in extended-spectrum -lactamase(ESBL)-producing Escherichia coli and Klebsiella pneumoniae have been reported in Korea. The aim of this study was to determine the nationwide prevalence of ESBL-producing E. coli and K. pneumoniae, and to investigate the types of ESBLs. METHODS: A total of 2,221 E. coli and 1,128 K. pneumoniae consecutive isolates were yearly collected from 12 hospitals in 1999 and 2000. ESBL production was performed by National Committee for Clinical Laboratory Standards methods and double synergy tests. The type of ESBL was determined by polymerase chain reaction (PCR), isoelectric focusing, and nucleotide sequence analysis. RESULTS: ESBL-producing E. coli and K. pneumoniae isolates were detected from all 12 hospitals participated. The proportion of ESBL-producers was 9.1%(2.0-19.6%) of the E. coli and 29.2% (10.0-60.8%) of the K. pneumoniae isolates. Among the 22 isolates sequenced, SHV-12 was found in six isolates, SHV-2a in three isolates, TEM-52 in five isolates, TEM-106 in three isolates, and each of TEM-15, TEM-20, TEM-43, and TEM-107 in one isolate. CTX-M-14 was also found in one isolate. CONCLUSION: ESBL-producing E. coli and K. pneumoniae are widespred to all levels of Korean hospitals. The most common types of ESBLs in Korea are SHV-12, SHV-2a, and TEM-52. In addition, we also identified new TEM-derived ESBLs.
Base Sequence ; Escherichia coli* ; Escherichia* ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Klebsiella* ; Korea* ; Pneumonia ; Polymerase Chain Reaction ; Prevalence*

In this work, the experimental conditions for two-dimensional gel electrophoresis of subretinal fluids (SRF) matrix metalloproteinases were established. The conditions tested included the composition of lysis solution and lysis method, the composition of rehydration solution and isoelectric focusing program (IEF), the composition of equilibration buffer and equilibration process and the composition of incubation solution and incubation methods. The main equipments used were IPGphor isoelectric focusing system from Amersham pharmacia and PROTEAN II xi cell from Bio-Rad, the gel strips used were the 18 cm long, pH 3 - 8 Linear immobiline DryStrips. Among the 9 samples analyzed, 2 were PVR-A, 2 were PVR-C1, 2 were PVR-C2, 2 were PVR-C3 and the remaining one could not be classified definitely. The new 2-DE MMPs method is better than Gelatin SDS-PAGE zymograhpy method, as it is higher in resolution, sensitivity and reproducibility. The experimental results suggested that the four types of MMPs expressed differently at different stages of PVR. Two of the MMPs isomers have same molecular weight (MW) but different in isoelectic points (pI). The four MMPs are determined to be MMP-1, MMP-2, MMP-9 and MMP-9, with MMP-9 has two active forms. In addition, MMP-9 and MMP-1 may be present in PVR-A samples but not in PVR-C samples, whereas MMP-2 is present in PVR-C but not in PVR-A samples. These results revealed the complex profiles of MMPs' expression in PVR. The new method can be applied to test MMPs expression in tissues, cells and other types of samples with a little modification in the protocol, and can be followed by mass spectroscopic analysis of MMPs.
Electrophoresis, Gel, Two-Dimensional ; Humans ; Isoelectric Focusing ; Matrix Metalloproteinases ; analysis ; Reproducibility of Results ; Retina ; enzymology

In the present study, isoelectronic focusing with different pH gradients (pH 3-5, 2-6) or migrating distances (8.5, 12 and 17 cm) and SDS-PAGE was used to separate continuous erythropoietin receptor activator (CERA), recombinant human erythropoietin (rhEPO), darbepoetin and endogenous EPO spiked in human urine with 37 degrees C overnight incubation. Double blotting and chemiluminescent visualization were used to detect the IEF and SDS-PAGE profiles. The bands of CERA profile were detected and well separated from the endogenous EPO and the other two EPO preparations with both SDS-PAGE and the IEF method using a gradient pH 3-5 and a migrating distance of 17 cm, and a significant particular band of CERA profile was found in the IEF result. These preliminary results indicated that the methods were reliable and reproducible for detecting CERA, and could be used as a routine procedure for anti-doping analysis.
Electrophoresis, Polyacrylamide Gel ; Erythropoietin ; urine ; Humans ; Isoelectric Focusing ; methods ; Polyethylene Glycols ; Recombinant Proteins

BACKGROUND: Acinetobacter baumannii is a glucose-nonfermenting gram-negative rod and is a well-recognized nosocomial pathogen. In recent years, A. baumannii strains showing resistance to carbapenems by producing metallo-beta-lactamases or OXA-type beta-lactamases have increased, and it is considered to be a serious clinical problem. But genotypes of carbapenemases produced by A. baumannii isolates in Korea have been rarely reported. The purpose of this study was to investigate the prevalence of imipenem-resistant A. baumannii and to determine the mechanism of resistance. METHODS: During the period of January through September, 2003, susceptibilities to imipenem of A. baumannii isolates from patients admitted in Kosin University Gospel Hospital in Busan, Korea were investigated. The modified Hodge and EDTA-disk synergy tests were performed for screening of carbapenemase and metallo-beta-lactamase-production. Minimal inhibitory concentrations (MICs) were determined by the agar dilution method. For detection of IMP, VIM and OXA-type beta-lactamases genes, polymerase chain reactions (PCR) were performed, and the DNA sequences of OXA-type beta-lactamases genes were determined by using the dideoxy-chain termination method. The isoelectric points of beta-lactamases were determined by isoelectric focusing. Pulsed-field gel electrophresis (PFGE) of the SmaI-digested genomic DNA was performed. RESULTS: A total of 193 strains of A. baumannii were collected from patients during the surveillance period. Twenty-seven percents (52/193) of A. baumannii isolates were resistant to imipenem. Among the 52 imipenem-resistant isolates, 41 isolates (78.8%) showed positive results in the modified Hodge test, but none of the isolates showed positive results in the EDTA-disk synergy test. Thirty-eight modified Hodge test-positive isolates harbored blaOXA-23 gene, but none of the isolates harbored IMP- or VIM-type metallo-beta-lactamases genes. Analytical isoelectric focusing revealed that all the 38 isolates had a nitrocefin-positive band at pI of 6.65. Thirty-five OXA-23-producing isolatesshowed a similar PFGE pattern when digested by SmaI endonuclease. CONCLUSION: Thirty-eight clinical isolates of A. baumannii acquired resistance to imipenem by producing OXA-23 beta-lactamase. Among them were 35 isolates thought to be originated from the same source, because they contained a similar chromosomal type. To the best of our knowledge, this is the first time that OXA-23 beta-lactamase has been detected in Korea.
Acinetobacter baumannii* ; Acinetobacter* ; Agar ; Base Sequence ; beta-Lactamases ; Busan* ; Carbapenems ; DNA ; Genotype ; Humans ; Imipenem ; Isoelectric Focusing ; Isoelectric Point ; Korea* ; Mass Screening ; Polymerase Chain Reaction ; Prevalence*

BACKGROUND: Increase in Escherichia coli and Klebsiella pneumoniae isolates with extended- spectrum beta-lactamase (ESBL) have been noted recently in Korea. Current NCCLS disk diffusion test is not sensitive to detect ESBL-producing strains. We have more isolates with intermediate or resistance to cefotaxime than to ceftazidime disk test. The aim of this study was to characterize the ESBLs produced by strains isolated from clinical specimens. METHODS: E. coli and K. pneumoniae strains with cefotaxime intermediate or resistance by disk method were tested for ESBL production by double-disk synergy, transfer of resistance by conjugation, and relative hydrolysis of beta-lactams and isoelectric point (pI) of cell sonicate. The types of beta-lactamase gene were determined by PCR. RESULTS: Nine of the 10 E. coli and all of the 18 K. pneumoniae strains tested were synergy test positive. The MIC of cefotaxime ranged from 1.5 to 400microgram/mL, with the inoculum of 10(4) CFU, but was much higher with larger inoculum. Some selected isolates showed that the resistance was transferable and the size of the plasmid was approximately 39 MDa. The MIC of cefotaxime was higher than that of was ceftazidime in majority of the transconjugants. The hydrolytic activity of the sonicate higher for cefotaxime than ceftazidime. TEM- and SHV-type genes were detected by PCR and the pI of the beta-lactamase was 5.9 for 4 TEM-type and 7.8 for one SHV-type. CONCLUSION: Majority of the cefotaxime intermediate or resistant E. coli and K. pneumoniae isolated from the tertiary care hospital are TEM- or SHV- type ESBL producers. The inoculum size significantly affects the MIC value of beta-lactams for the ESBL-producing strains. The ESBL hydrolyzes cefotaxime more actively than ceftazidime, and the ESBL gene is transferable by conjugation.
beta-Lactamases ; beta-Lactams ; Cefotaxime ; Ceftazidime ; Diffusion ; Escherichia coli* ; Escherichia* ; Hydrolysis ; Isoelectric Focusing ; Isoelectric Point ; Klebsiella pneumoniae* ; Klebsiella* ; Korea ; Plasmids ; Pneumonia ; Polymerase Chain Reaction ; Tertiary Healthcare