Glanzmann's thrombasthenia is a rare autosomal recessive hemorrhagic disorder characterized by prolonged bleeding time, ad deficient or absent clot retraction in the presence of normal platelet count. The major underlying abnormality in this disease is grossly defective first-phase aggregation of platelet, which are unresponsive to ADP or other platelet agonists such as epinephrine, collagen, thrombin in any concentration. This disability is caused by a decrease or absence of the platelet membrans glycoprotein IIb-IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix fibronectin, and vitronectin On the development of surface labeling technique, a variety of biochemical techniques such as radioimmunoassay, crossed immunoelectrophoresis and SDS-PAGE have been used to study the structure and the function of platelet membrane glycoproteins, and to detect the platelet functional defect. But all of these techniques demand a relatively large amount of homogeneous paletelet population that requires manipulation through isolation and washing procedures before analysis. In order to eliminaste such an intricate procedure, we have applied method for analyzing platelet surface components in whole blood using monoclonal antibody and flow cytometry to recognize the absence of severe reduction of platelet membrane glycoprotien llb-llla complex. Platelet analysis by flow cytometry is a successful alternative rapid diagnostic technique for Glanzmann's thrombasthenia patients as well as well as for carriers of this disease. Fow cytometry technique provides a sensitive tool for investigating platelet functional defects caused by altered expression or deficiency of platelet surface proteins.
Adenosine Diphosphate ; Adhesives ; Bleeding Time ; Blood Platelets* ; Clot Retraction ; Collagen ; Electrophoresis, Polyacrylamide Gel ; Epinephrine ; Fibronectins ; Flow Cytometry* ; Glycoproteins ; Hemorrhagic Disorders ; Humans ; Immunoelectrophoresis, Two-Dimensional ; Membrane Glycoproteins* ; Membrane Proteins ; Membranes* ; Platelet Count ; Platelet Membrane Glycoproteins ; Radioimmunoassay ; Thrombasthenia* ; Thrombin ; Vitronectin

Adult ; Blood Glucose ; analysis ; Diabetes Mellitus ; blood ; Electrophoresis, Agar Gel ; Electrophoresis, Paper ; Female ; Hemoglobin A ; analysis ; Humans ; Male

OBJECTIVETo evaluate the effects of two sample preparation methods on two-dimensional polyacrylamide gel electrophoresis (2-DE)-based serum protein separation, and produce high-resolution and reproducible 2-DE images for identifying disease-related serum protein.

METHODSDirect solubilization and hot SDS methods were used separately to extract and handle the total proteins of serum samples from patients with colorectal carcinoma. Immobilized pH gradient 2-DE was used to separate the total proteins. After image analysis of silver-stained 2-D gels, 3 differential protein spots were identified by matrix-assisted laser desorption/time-of-flight mass spectrometry.

RESULTSThe total proteins treated with hot SDS method were used to perform 2-DE. 2-DE patterns with high resolution and reproducibility were obtained for human serum samples. 2-DE was performed 3 times for the samples treated by direct solubilization and hot SDS methods, respectively, resulting in the average number of spots of 675-/+46 and 702-/+49, respectively. The average matching protein spots were 573-/+42 and 623-/+52, with average matching rate of 85.3% and 89.6%, respectively. The average position deviation of matched spots in different gels was 0.85-/+0.30 mm and 0.81-/+0.28 mm in IEF direction, and 1.02-/+0.18 mm and 0.97-/+0.12 mm in SDS-PAGE direction. Mass spectrometry of the 2-D gels treated with hot SDS method generated high-quality mass spectra, and the sample preparation method allowed detection of relatively low abundance protein.

CONCLUSIONHot SDS method is more effective for human serum protein sample preparation and well-resolved, reproducible 2-DE profiles of human serum have been established in this study.

Blood Protein Electrophoresis ; Blood Proteins ; analysis ; isolation & purification ; Electrophoresis, Gel, Two-Dimensional ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Peptide Mapping ; Protein Interaction Mapping ; Reproducibility of Results

BACKGROUND: The importance of extended-spectrum beta-lactamases (ESBL) produced in gramnegative bacilli is now well recognized, but most clinical laboratories have problems in detecting and interpreting ESBL and implicating the findings in nosocomial infections caused by ESBL producing gram-negative bacilli. The present study aims primarily to evaluate the distributions of these enzymes among Escherichia coli and Klebsiella pneumoniae, the most frequent isolates of Enterobacteriaceae producing ESBL, to differentiate the types of enzymes in theses isolates and finally to relate the clonality of specific types within a part of Daegu city. METHODS: The clinical isolates of 1, 242 E. coli and 859 K. pneumoniae were screened for ESBL production by the disk diffusion method of the National Committee of Clinical Laboratory Standard, and it was confirmed by the double-disk synergy test (DDS). Antimicrobial susceptibility test was performed by the agar dilution method. The presence of -lactamase was tested by polymerase chain reaction (PCR) and plasmid analysis. Isoelectric focusing and nucleotide sequence analysis were performed to evaluate ESBL types. Pulsed-field gel electrophoresis (PFGE) of XbaI-digested DNA fragments was carried out to determine the extend of clonality within the hospital. RESULTS: Of 34 isolates of E. coli and 31 isolates of K. pneumoniae ramdomly selected from those isolates screened for ESBL production were further tested by DDS to confirm its production: 30 (88.2%) E. coli and 29 (93.5%) K. pneumoniae were positive. TEM-52 and SHV-12 were present both in E. coli and K. pneumoniae, but SHV-2a was distributed only in K. pneumoniae. The resistance was transferable in 66.7% of E. coli and 68.9% of K. pneumoniae. Six and 5 PFGE patterns were shown by E. coli and K. pneumoniae, respectively. Among the 5 patterns of K. pneumoniae, type B was dominant, suggesting a clonal outbreak in the hospital. CONCLUSIONS: The ESBL specific enzyme types were TEM-52, SHV-2a and SHV-12. Despite many different PFGE patterns of the ESBL producing isolates, a few outbreak and edemic clones appear to be prevalent in Dongsan Medical Center.
Agar ; Base Sequence ; beta-Lactamases* ; Clone Cells ; Cross Infection ; Daegu ; Diffusion ; DNA ; Electrophoresis, Gel, Pulsed-Field ; Enterobacteriaceae ; Escherichia coli* ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Plasmids ; Pneumonia ; Polymerase Chain Reaction

OBJECTIVETo analyse clinical feasibility of two electrophoresis procedures of seminal plasma proteins, agarose gel electrophoresis and SDS-agarose gel electrophoresis.

METHODSSixty-nine semen samples were examined and classified into three groups: the asthenozoospermia (n = 22), the asthenoteratozoospermia (n = 19), and the relative normal group (n = 28) with normal routine and special test results, according to WHO routine and special test criterion. Then, the seminal plasma protenis were separated by two different electrophoresis, with SDS-agarose and agarose support medium, the buffer pH 7.0 and 9.2 respectively. The agarose gel electrophoresis was done under various sample loading time, motion power and staining modules. The completed gels were scanned and compared the each other statistically.

RESULTSSeminal plasma proteins can be separated into 4 strips by SDS-agarose gel electrophoresis with acid crystal violet, and the strips were diffusion and with dark background. However, 6 clear strips named A, B, C, D, E, and F can be obtained by agarose gel electrophoresis with 6 min. After samples were loaded and stained by amidoblack, there showed appropriate spaces among strips, and it was very easy to scan the drying gel by a densitometer. Using agarose gel electrophoresis, the statistical difference in strip C and E was shown between the asthenozoospermia and the relative normal group, and between the asthenozoospermia and the asthenoteratozoospermia, however, not between the relative normal and the asthenoteratozoospermia group. Moreover, the samples in the relative normal group with normal routine and special test results were in different electrophoresis maps.

CONCLUSIONAgarose gel electrophoresis of seminal plasma proteins with buffer pH 9.2, 6 min. sample loading and amidoblack stain was a simple, fast and fit technique for clinic.

Adult ; Electrophoresis, Agar Gel ; methods ; Electrophoresis, Polyacrylamide Gel ; methods ; Humans ; Male ; Proteins ; analysis ; Semen ; chemistry ; Staining and Labeling

BACKGROUND: ESBL-producing Klebsiella spp. are increasing worldwide, and infections with ESBL-producing organisms are usually hospital-acquired and common infections with ESBL-producing organisms include urinary tract infections, peritonitis, cholangitis, intraabdominal abscesses and nosocomial pneumonia. We studied the phenotypic and genotypic characteristics of ESBL-producing K. pneumoniae, which were isolated from the patients in St. Vincent Hospital. METHODS: Susceptibility to antibiotic was determined by standard disk diffusion and agar dilution methods for all 22 strains of K. pneumoniae. PCR amplifications were performed with primers specific for TEM. SHY, and CMY-1 genes, and the DNA of the amplified products were sequenced. Total DNA was extracted from the isolates restricted with XbaI, and fingerprinted using pulsed-field gel electrophoresis. Crude preparations of beta-lactamase were obtained by sonications and used for characterization of beta-lactamase by isoelectric focusing. RESULTS: The MIC90 values for ceftazidime and aztreonam were 128 microgram/mL and 64 microgram/mL, respectively. The MIC90 values for cefotaxime and sulperazon were 8 microgram/mL and 4microgram/mL, respectively, and that for cefoxitin was 1.0 microgram/mL, which is much lower than the value for third-generation cephalosporins. The MICs for ciprofloxacin. cefepime, and imipenem were less than 1 microgram/mL for all organisms, which is within the susceptible range. Isoelectric focusing studies demonstrated three beta-lactamases with pls of 5.. (TEM-1), 7.6 (SHV-2a), and 8.2 (SHV-12). The presence of blaSHV and blaTEM genes was confirmed by specific PCRs and DNA sequencing analysis. but blaCMY-1 genes were not found. According to DNA sequencing analysis, 21 K. pneumoniae strains produced SHV-12 ESBL and one strain produced SHV-2a ESBL. CONCLUSIONS: Our results suggest that the resistance of K. pneumoniae from clinical isolates to extended spectrum beta-lactam antibiotics may be due to the production of SHY-type ESBL. This approach in detecting and characterizing ESBL will be valuable for the control of infection and antibiotics use in medical institution.
Abscess ; Agar ; Anti-Bacterial Agents ; Aztreonam ; beta-Lactamases ; Cefotaxime ; Cefoxitin ; Ceftazidime ; Cephalosporins ; Cholangitis ; Ciprofloxacin ; Dermatoglyphics ; Diffusion ; DNA ; Electrophoresis, Gel, Pulsed-Field ; Humans ; Imipenem ; Isoelectric Focusing ; Klebsiella pneumoniae* ; Klebsiella* ; Peritonitis ; Pneumonia ; Polymerase Chain Reaction ; Sequence Analysis, DNA ; Sonication ; Urinary Tract Infections

The biological interpretation of two-dimensional (2D) gel electrophoresis experiments is a key step toward understanding the functions of biological systems. We here present a web-based integrated database, called 2DSpotDB, for the management of proteome data derived from several pathogens. The 2DSpotDB was established as a part of the management of a pathogen proteome project at the Korea National Institute of Health. The goals of the 2DSpotDB implementation are to store and define important pathogen genes, retrieve information obtained by 2D polyacrylamide gel electrophoresis and mass spectrometry, and create an integrated system to provide pathogen proteome information for biological scientists. This database currently contains 14 gels and information on 387 protein spots, among which 329 proteins were identified and annotated.
Acrylic Resins ; Data Mining ; Electrophoresis ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Gels ; Korea ; Mass Spectrometry ; Proteins ; Proteome

We investigated the biochemiesl change of keratin by the methods of SDS-PAGE and Two-dimensional gel electrophoresis, and observed electron microscopic ultrestructural changes in five Unna-Thost palmoplantar keratoderma patients and two normal adults. The results are summarized as follows : 1. The increased bands of 51 kd and newly appearing 48 kd, 56 kd keratins were observed on the SDS-PAGE and compared to the normsl control. 2. The newly appearing 48 kd(acidic) paired with 56 kd(basic) keratins and 51 kd keratin and the disappearance of 59 kd(basic), 64 kd(basic) keratins were observed on the two-dimensional gel electrophoresis and compared to the normal control. 3. The variable sized, numerous, globular, irregularly beam-shaped and granular kerstohyaline granules were scattered in the granular cell and corneocyte. Numerous ribosomes were noted between the clumped tonofibrils and around the keratohyaline granules. The lipid droplets were seen in the corneocytes and granular cells on the electron microscope.
Adult ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Humans ; Keratoderma, Palmoplantar* ; Ribosomes

In this study,the protein in different Cordyceps samples,which include fresh sample( S1),22 ℃ drying sample( S2),37 ℃ drying sample( S3) and 60 ℃ drying sample( S4),were analyzed by sodium dodecylsupinate-polyacrylamide gel electrophoresis( SDS-PAGE) and two-dimensional electrophoresis( 2-DE). The total protein contents in Cordyceps samples were from 1. 655-4. 493 mg·g~(-1) and the protein contents in fresh sample was the highest. The results of SDS-PAGE showed that the mainly ranges of protein molecular weight of Cordyces samples were 10-100 kDa and the numbers of protein bands were 28 to 41,the fresh sample had the maximum number of protein bands. The 2-DE profiles were analyzed by PDQuest software. The resulted indicated that 488-876 protein spots were detected in different Cordyceps samples and the isoelectric point( pI) was distributed between 4. 5 and 6. 5,the protein molecular weight was distributed in 10-20 kDa and 25-100 kDa,the fresh sample had the maximum number of protein spots. Therefore,the drying process could decrease contents and species of protein in Cordyceps,and the different drying conditions had different effects on protein. These results provide a reference for improving the drying process of Cordyceps.
Cordyceps ; chemistry ; Desiccation ; methods ; Electrophoresis, Gel, Two-Dimensional ; Electrophoresis, Polyacrylamide Gel ; Fungal Proteins ; analysis ; Molecular Weight

BACKGROUND: Recently biphasic agarose gel electrophoresis method using Chol/Trig ComboTM for simultaneous detection of cholesterol and triglyceride on lipoprotein fractions has been developed to facilitate the classification and interpretation of abnormal lipoprotein patterns of patients with hyperlipidemia. The author analysed serum lipid profiles using Chol/Trig ComboTM in the patients of OPD in Department of Internal Medicine. METHODS: Measurement of serum cholesterol and triglyceride using enzymatic method was performed in the sera of 415 patients from April, 2006 to July, 2006. Simultaneously, we electrophoresed serum cholesterol and triglyceride using Chol/Trig ComboTM with analysis software (ED BANK, Helena Laboratories, Saitama, Japan). RESULTS: According to ATP III guideline, we set up standard cholesterol as 200 mg/dL and triglyceride as 150 mg/dL, respectively, and the patients were classified into control, hypercholesterolemic, hypertriglyceridemic and hypercholesterolemic/hypertriglyceridemic groups, respectively. Hypercholesterolemic group had higher HDL and LDL, hypertriglyceridemic group had higher HDL, VLDL and the rates of positive modified LDL, hypercholesterolemic/hypertriglyceridemic group have higher VLDL and the rates of positive modified LDL than the control. Otherwise, the rates of positive modified LDL were 19.8% in the control, 23.6% in the hypercholesterolemic group, 50.9% in hypertriglyceridemic group, and 39.3% in hypercholesterolemic/hypertriglyceridemic group. CONCLUSIONS: The frequency of the rates of positive modified LDL and VLDL was higher in the hypertriglyceridemic group than those in the control.
Adenosine Triphosphate ; Cholesterol ; Electrophoresis ; Electrophoresis, Agar Gel ; Humans ; Hyperlipidemias ; Lipoproteins