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

In order to understand the protein functions that are related to disease, it is important to detect the correlation between amino acid mutations and isease. Many mutation studies about disease-related proteins have been carried out through molecular biology techniques, such as vector design, protein engineering, and protein crystallization. However, experimental protein mutation studies are time-consuming, be it in vivo or in vitro. We therefore performed a bioinformatic analysis of known disease-related mutations and their protein structure changes in order to analyze the correlation between mutation and disease. For this study, we selected 111 diseases that were related to 175 proteins from the PDB database and 710 mutations that were found in the protein structures. The mutations were acquired from the Human Gene Mutation Database (HGMD). We selected point mutations, excluding only insertions or deletions, for detecting structural changes. To detect a structural change by mutation, we analyzed not only the structural properties (distance of pocket and mutation, pocket size, surface size, and stability), but also the physico-chemical properties (weight, instability, isoelectric point (IEP), and GRAVY score) for the 710 mutations. We detected that the distance between the pocket and disease-related mutation lay within 20 A (98.5%, 700 proteins). We found that there was no significant correlation between structural stability and disease-causing mutations or between hydrophobicity changes and critical mutations. For large-scale mutational analysis of disease-causing mutations, our bioinformatics approach, using 710 structural mutations, called "Structural Mutatomics," can help researchers to detect disease-specific mutations and to understand the biological functions of disease-related proteins.
Computational Biology ; Crystallization ; Humans ; Hydrophobic and Hydrophilic Interactions ; Isoelectric Point ; Molecular Biology ; Point Mutation ; Protein Engineering ; Proteins

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: 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

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.
Acinetobacter baumannii* ; Acinetobacter* ; Agar ; beta-Lactamases ; Busan ; Clinical Coding ; Consensus ; Humans ; Imipenem ; Isoelectric Focusing ; Isoelectric Point ; Korea ; Microbial Sensitivity Tests ; Molecular Epidemiology* ; Polymerase Chain Reaction ; Prevalence

OBJECTIVESTo analyze human spermatozoa membrane proteins by two-dimensional gel electrophoresis and to provide a basis for drawing the protein map of normal human spermatozoa membrane proteins.

METHODSSpermatozoa were purified by Percoll density centrifugation, and spermatozoa membrane proteins were analyzed by two-dimensional gel electrophoresis using isoelectric focusing and polyacrylamide gel electrophoresis.

RESULTSAbout 800 protein spots could be identified by the imaging analysis system. The molecular weight and isoelectric point of most proteins were 20,000 to 100,000 and 3.0 to 7.0 respectively.

CONCLUSIONSThere are more than 800 types of proteins in the human spermatozoa membrane. The spermatozoa membrane protein can be identified with a certain precision by two-dimensional gel electrophoresis.

Electrophoresis, Gel, Two-Dimensional ; Humans ; Image Processing, Computer-Assisted ; Isoelectric Focusing ; Isoelectric Point ; Male ; Membrane Proteins ; chemistry ; Molecular Weight ; Spermatozoa ; chemistry

A 36-year-old pregnant woman with gestational diabetes mellitus and anemia was found to have an abnormal Hb (comprising 18.7%) in the automated midget low pressure cation- exchange chromatography (DiaSTATTM, Bio-Rad, USA) for Hb A1c assay. The abnormal Hb revealed an abnormal peak emerged slightly later than normal Hb A1 in DiaSTATTM chromatogram, subsequently confirmed by cellulose acetate membrane electrophoresis and isoelectric focusing. This hemoglobinopathy with high isoelectric point was noted and abnormal chain globin was prepared by chromatography. Family study was carried out and this chain variant was also found in four other family members, and all of them had no clinical abnormalities, except well controlled diabetes. As the results from peptide mapping, amino acid analysis and sequencing, abnormal Hb of the patient was finally identified as Hb Queens[ 34 (B15)Leu-->Arg] without clinical abnormalities.
Adult ; Anemia ; Cellulose ; Chromatography ; Diabetes, Gestational ; Electrophoresis ; Female ; Globins ; Hemoglobin A, Glycosylated ; Hemoglobinopathies ; Humans ; Isoelectric Focusing ; Isoelectric Point ; Membranes ; Peptide Mapping ; Pregnancy ; Pregnant Women

OBJECTIVETo explore the feasibility of membrane protein-based two-dimensional electrophoresis (2-DE) in the investigations of chondrocyte-related diseases and its complementarity with total protein-based 2-DE.

METHODSKnee cartilage samples were obtained to isolate the chondrocytes with type II collagenase/hyaluronidase digestion. The membrane proteins and total proteins were extracted and loaded separately onto PH3.0-10.0 non-linear gel strip for 2-DE analysis. The qualities of membrane protein-based 2-DE gels were evaluated, and the difference between the distribution profiles of the membrane protein spots and that of the total protein were observed and their complementarities were evaluated.

RESULTSMembrane protein-based 2-DE generated high-quality gel images, and on each gel 412.3±13.5 protein spots were identified. These spots were distributed in the region of isoelectric point pH 5.0-9.0. In the gel images generated by the total proteins, 564.3±5.9 protein spots were identified in each image, and the spots were distributed in the region of isoelectric point pH 3.0-7.0.

CONCLUSIONMembrane protein-based 2-DE of the chondrocytes can generate high-quality gel images, and the isoelectric distribution of the protein spots is complementary to that of total protein, which provides valuable information for chondrocyte-related diseases.

Adult ; Chondrocytes ; chemistry ; Electrophoresis, Gel, Two-Dimensional ; methods ; Female ; Humans ; Isoelectric Point ; Male ; Membrane Proteins ; isolation & purification ; Middle Aged

BACKGROUND: In recent years, Acinetobacter baumannii isolates acquired resistance to cefepime have increased significantly. The aim of this study was to survey the prevalence of PER-1 extendedspectrum beta -lactamase (ESBL)-producing A. baumannii isolates in a University Hospital, Busan, Korea. METHODS: Antimicrobial susceptibilities were tested by the disk diffusion method, and double disk synergy test was performed for screening of ESBL-production. MICs were determined by agar dilution method. The isoelectric points of beta -lactamases were determined by isoelectric focusing. Transferability of cefepime-resistance were tested by conjugation. blaPER-1 and blaPER-2 alleles were detected by PCR, and the DNA sequences of amplified products were determined by using the dideoxy-chain termination method. RESULTS: Among 51 clinical isolates of A. baumannii intermediate or resistant to cefepime, 10 isolates (19.6%) showed positive results in double disk synergy test. PCR-based experiments detected blaPER-1 gene in all the 10 isolates. All the isolates contained three beta -lactamase bands: pI 5.3, 7.9, and 9.4. MICs of ampicillin, piperacillin, cephalothin, cefoxitin, cefoperazone, ceftazidime, cefotaxime, cefepime, and aztreonam were >256 mg/L, respectively, and them of imipenem were 8-16 mg/L. CONCLUSION: The prevalence of PER-1-producing A. baumannii strains in Busan was less than that of in Seoul. But an outbreak of infection caused by this strain in an intensive care unit shows that spread of PER-1-producing A. baumannii strains can be anticipated in a near future. Prevention of hospital infection by these resistant microorganisms are needed.
Acinetobacter baumannii* ; Acinetobacter* ; Agar ; Alleles ; Ampicillin ; Aztreonam ; Base Sequence ; Busan* ; Cefoperazone ; Cefotaxime ; Cefoxitin ; Ceftazidime ; Cephalothin ; Cross Infection ; Diffusion ; Imipenem ; Intensive Care Units ; Isoelectric Focusing ; Isoelectric Point ; Korea* ; Mass Screening ; Piperacillin ; Polymerase Chain Reaction ; Prevalence* ; Seoul