OBJECTIVETo search for a new method of screening for molecular targets for androgen-dependent prostate cancer.

METHODSWe collected tissue samples and paired serum samples from 3 cases of androgen-dependent prostate cancer (ADPC) treated by surgical resection, and included another 3 samples of benign prostatic hyperplasia (BPH) tissue and normal human serum in the control group. The total proteins extracted were separated and transmembrane by two-dimensional gel electrophoresis, followed by hybridization with the sera of the patients with ADPC and those with hormone-independent prostate cancer (HIPC) as the primary antibodies. The differentially expressed proteins were compared by Western blot, analyzed by MALDI-TOF-MS mass spectrography, and verified by RT-PCR and Western blot following bioinformatic identification.

RESULTSThis modified method exhibited a significantly better effect in displaying differentially expressed proteins, by which 12 differentially expressed protein spots were identified, including Beclin1, glutathione S-transferase P (GSTP1-1), ZBTB7, dihydrodiol dehydrogenase 2 (DDH), enolase (ENO1), glucose-dependent insulin-releasing peptide receptor (GIPR), Mn-superoxide dismutase (MnSOD), phosphoglycerate mutase 1 (PGAM1), amino-peptidyl-prolyl cistrons isomerase (PPIA), and phospholipid-PE-binding protein (PEBP). The mRNA and protein expressions of Beclin1 were significantly down-regulated in androgen-dependent prostate cancer tissues.

CONCLUSIONThis modified serum-guided immunoblotting technique has provided a new method for clarifying the molecular mechanisms of the occurrence and progression of HIPC, in which Beclin1-mediated autophagy may play a key role.

Biomarkers, Tumor ; blood ; Blotting, Western ; Humans ; Immunoblotting ; methods ; Male ; Mass Spectrometry ; Prostatic Neoplasms ; genetics ; metabolism ; Proteomics

AIMTo detect the expression of a novel protein, hemangiopoietin (HAPO), in the human fetal liver at the protein level.

METHODSMonoclonal antibodies (moAbs) against HAPO were produced by traditional hybridoma technique. Their affinities were calculated from the results of non-competitive ELISA and the antibodies were purified by protein G affinity chromatography. Expression of HAPO in the liver was detected by SDS-PAGE and Western blot.

RESULTSFive strains of moAbs were screened out in total, among which three were IgG1 and the other two were IgM. Their light trains were all belonged to kappa. The relative affinities of the three IgG1 form moAbs were 3.06 x 10(9) mol/L, 6.07 x 10(8) mol/L and 1.71 x 10(10) mol/L respectively. After purification, the purity of the moAbs could reach more than 99%. HAPO expression was detected at the protein level in the human fetal liver, and the apparent molecular weight of the nature HAPO was very close to but a little higher than our recombinant one.

CONCLUSIONHAPO was expressed in the human fetal liver at the protein level.

Antibodies, Monoclonal ; Blotting, Western ; Fetus ; Humans ; Immunoblotting ; Liver ; embryology ; metabolism ; Proteoglycans ; metabolism

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 AND OBJECTIVES: The purpose of this study was to subculture normal human middle ear epithelial (NHMEE) cells, investigate whether the subcultured NHMEE cells could have ability to differentiate into secretory cells, and establish a method to get cultured NHMEE cells for further study of human middle ear epithelial differentiation and secretion. MATERIALS AND METHOD: Freshly isolated epithelial cells from healthy middle ear mucosa were subcultured repeatedly after enzymatic disaggregation in serum-free medium on plastic tissue culture dishes. The subcultured cells were counted after every passage and tested for secretory differentiation in air-liquid interface (ALI) cultures. The apical secretion of cultured NHMEE cells were characterized by immunoblotting and Western blotting. RESULTS: Attachment rate of subcultured NHMEE cells was over 70% through every passage. Cells proliferated by 22 fold from passage-1 to passage-2 (P-2), but passage-4 cells did not proliferate. P-2 NHMEE cells in ALI cultures was stained with mucin antibody (H6C5) but not b-tubulin antibody. Cultured NHMEE cells secreted mucin and lysozyme. CONCLUSION: P-2 NHMEE cell cultures retained many important features of normal epithelium and were suitable for conducting many studies of human middle ear epithelial cell biology including cell differentiation and secretion.
Biology ; Blotting, Western ; Cell Culture Techniques ; Cell Differentiation ; Ear, Middle* ; Epithelial Cells ; Epithelium ; Humans* ; Immunoblotting ; Mucins ; Mucous Membrane ; Muramidase ; Plastics

BACKGROUND AND OBJECTIVES: The purpose of this study was to subculture normal human nasal epithelial (NHNE)cells without compromising their ability to differentiate into secretory and ciliated cells and to study the effect of retinoic acid (RA)on mucous and serous secretions in each passaged cells. MATERIALS AND METHOD: Freshly isolated nasal epithelial cells from normal inferior turbinates were subcultured repeatedly in serum-free medium on plastic tissue culture dishes. The subcultured cells were tested after every passage for secretory differentiation in air-liquid interface (ALI) cultures. The apical secretion of cultured NHNE cells was characterized by immunoblotting and Western blotting. RESULTS: Cultured NHNE cells secreted mucin and lysozyme. RA was essential for mucociliary and secretory differentiation. The epithelium became squamous and mucin secretion decreased when RA was deleted from the culture media. Cells from passage 1(P-1) through passage-2 (P-2) remained competent to differentiate into mucous and squamous cells when grown in air-liquid interface culture. CONCLUSION: P-2 NHNE cell cultures retained many important features of normal epithelium and were suitable for conducting many studies of upper airway cell biology with an expanded cell pool.
Blotting, Western ; Cell Culture Techniques ; Culture Media ; Epithelial Cells ; Epithelium ; Humans* ; Immunoblotting ; Mucins ; Muramidase ; Plastics ; Tretinoin ; Turbinates

OBJECTIVETo research on the substantial foundation of the medical speciality of Chinese traditional medicines from immunogenicity.

METHODControl antigen with hot nature was prepared from the mixture of the aqueous extracts of three Chinese traditional medicines with three typical hot nature of Alpinia officinarum, Cinnamomum cassia and Curculigo orchioides, while that with cold nature prepared with Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, and polyclonal antibody was prepared by immunizing rabbit with control antigen. Dot blotting was performed between the polyclonal antibody of control antigen and the aqueous extracts of nine Chinese traditional medicines on a piece of PVDF membrane, and the blotting signals were analyzed by the software of Quantity One.

RESULTBlotting signals with hot control antigen of nine Chinese traditional medicines in descending were Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum and Phellodendron chinense, which degree of similarity to control antigen in peak value were 57.33%, 43.56 %, 34.16%, 30.2%, 28.81%, 26.53%, 21.68%, 17.62% and 14.85%, respectively. Blotting signals with cold control antigen were Rheum palmatum, Anemarrhena asphodeloides, Coptis chinensis, Phellodendron chinense, Zingiber officinale, Lonicera japonica, Fraxinus rhynchophylla, Eucommia ulmoides and Aconitum carmichaeli in descending, of which degree of similarity to cold control antigen in peak value were 55.22%, 54.23%, 46.72%, 34.08%, 30.3%, 24.48%, 24.33%, 20.35% and 15.17%, respectively. Results of cluster analysis with Wistar's method showed that nine medicines were classified into two groups, one group included Phellodendron chinense, Anemarrhena asphodeloides, Coptis chinensis, Rheum palmatum, another was Zingiber officinale, Aconitum carmichaeli, Eucommia ulmoides, Fraxinus rhynchophylla, Lonicera japonica.

CONCLUSIONBlotting signals of nine medicines with control antigen regularly varied with the alteration of medicine nature. The more similarity degree of the tested medicine to control antigen was smaller, the more distance of the tested medicine to control antigen was further. Dot immunoblotting was a practical and effective new method in researching the substantial foundation of the medical speciality of Chinese traditional medicines.

Animals ; Antibodies, Monoclonal ; immunology ; Antibody Formation ; Antigens ; immunology ; Blotting, Western ; Cold Temperature ; Drugs, Chinese Herbal ; analysis ; chemistry ; classification ; Hot Temperature ; Immunoblotting ; methods ; Male ; Medicine, Chinese Traditional ; methods ; Rabbits

OBJECTIVES: The purpose of this study was to evaluate the anti-cancer activity of cisplatin by studying its effects on cell viability and identifying the mechanisms underlying the induction of cell cycle arrest and apoptosis on oral squamous cell carcinoma (OSCC) cell lines with varying p53 mutation status. MATERIALS AND METHODS: Three OSCC cell lines, YD-8 (p53 point mutation), YD-9 (p53 wild type), and YD-38 (p53 deletion) were used. To determine the cytotoxic effect of cisplatin, MTS assay was performed. The cell cycle alteration and apoptosis were analyzed using flow cytometry. Western blot analysis was used to detect the expression of cell cycle alteration- or apoptosis-related proteins as well as p53. RESULTS: Cisplatin showed a time- and dose-dependent anti-proliferative effect in all cell lines. Cisplatin induced G2/M cell accumulation in the three cell lines after treatment with 0.5 and 1.0 µg/mL of cisplatin for 48 hours. The proportion of annexin V-FITC-stained cells increased following treatment with cisplatin. The apoptotic proportion was lower in the YD-38 cell line than in the YD-9 or YD-8 cell lines. Also, immunoblotting analysis indicated that p53 and p21 were detected only in YD-8 and YD-9 cell lines after cisplatin treatment. CONCLUSION: In this study, cisplatin showed anti-cancer effects via G2/M phase arrest and apoptosis, with some difference among OSCC cell lines. The mutation status of p53 might have influenced the difference observed among cell lines. Further studies on p53 mutation status are needed to understand the biological behavior and characteristics of OSCCs and to establish appropriate treatment.
Apoptosis ; Blotting, Western ; Carcinoma, Squamous Cell* ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Line ; Cell Survival ; Cisplatin* ; Epithelial Cells* ; Flow Cytometry ; Immunoblotting

No abstract available.
Antibodies* ; Borrelia burgdorferi* ; Borrelia* ; Immunoblotting* ; Lyme Disease*

OBJECTIVETo assess the prevalence of anti-alpha-enolase antibody in systemic autoimmune diseases in Chinese patients and its role in endothelial cell apoptosis.

METHODSThe reactivity of anti-alpha-enolase antibody in a variety of autoimmune disorders in Chinese patients was evaluated by dot blot assay. Endothelial cell apoptosis was investigated by in vitro incubation of endothelial cells with IgG purified from anti-alpha-enolase antibody-positive sera, with or without pre-incubation with recombinant alpha-enolase.

RESULTSAnti-alpha-enolase antibody was prevalent in different systemic autoimmune diseases with relatively high reactivity in Chinese patients. In vitro incubation of endothelial cells with IgG containing anti-alpha-enolase antibody induced apoptosis in a time- and dose-dependent manner. Apoptosis was partly inhibited by pre-incubation of the endothelial cells with recombinant alpha-enolase.

CONCLUSIONSOur data suggest that alpha-enolase is a common auto-antigen recognized by anti-endothelial cell antibodies in connective tissue disease. Interaction between alpha-enolase and its autoantibody plays a role in endothelial cell apoptosis. Changes other than cell killing may contribute to the pathogenesis of endothelial damage and microvascular lesions.

Adolescent ; Adult ; Apoptosis ; drug effects ; Autoantibodies ; immunology ; pharmacology ; Autoimmune Diseases ; immunology ; Blotting, Western ; Cell Line ; Endothelial Cells ; cytology ; drug effects ; Female ; Flow Cytometry ; Humans ; Immunoblotting ; Male ; Middle Aged ; Phosphopyruvate Hydratase ; immunology ; metabolism ; Young Adult

BACKGROUND: Contractile dysfunction of rat myocardium in postinfarction remodeling is characterized by decreased response of myofilament both to calcium(Ca++) and adrenergic stimuli. This study tested the hypothesis that above alterations may be linked to molecular switches among the isoforms of troponin T and troponin I, the major regulators of myocardial responsiveness. METHODS: Using Sprague-Dawley rat as a model, myocardiums of fetal heart, 1 day postnatal heart, normal adult heart(n=4), and non-infarcted area of postinfarction heart(n=4, 3 months after left coronary artery ligation, mean LVEDP=21.4mmHg) were studied. For the detection on molecular switches, western blotting and semiquantitative RT-PCR were employed. RESULTS: Immunoblotting of rat myocardium showed normal developmental isoform switch of troponin protein. There was distinct isoform patterns specific for postinfarction rat heart. The postinfarction myocardium developed a marked increase in the fetal isoform and marked decrease in the adult isoform of troponin T, resulting in the reversed ratio of fetal/adult cardiac troponin T isoform(normal adult rat=0.60+/-0.1 vs postinfarction rat=1.79+/-0.15, p<0.01). Also, the postinfarction heart showed approximately 20% decrease in the amount of adult troponin I isoform. In RT-PCR experiments, the postinfarction hearts were characterized by increased amplification of fetal troponin R isoform cDNA(fetal troponin R/GAPDH ; normal adult rat=0.22, postinfarction rat=0.84). However, there was no siginificant difference in the amplification of troponin I cDNA between normal and postinfarction heart. CONCLUSION: These experimental findings indicate that there are molecular switches operative among the regulatory protein troponin T and I in the rat myocardium with development of postianfarction heart failure.
Adult ; Animals ; Blotting, Western ; Coronary Vessels ; DNA, Complementary ; Fetal Heart ; Heart ; Heart Failure ; Humans ; Immunoblotting ; Ligation ; Myocardial Infarction* ; Myocardium* ; Myofibrils ; Protein Isoforms* ; Rats* ; Rats, Sprague-Dawley ; Troponin I ; Troponin T* ; Troponin*