BACKGROUND: HLA-DR typing kits using reverse-SSO (sequence specific oligonucleotide) method show considerable ambiguities in HLA-DRB1 generic typing. We analyzed the ambiguities of the Dynal RELI(TM) SSO HLA-DRB test (Dynal DRB test) and developed an 'Interpretation Program for Koreans'. METHODS: A total of 3,000 Koreans were typed for HLA-DRB1/B3/B4/B5 using the 36 probe Dynal DRB test and all of the cases showing ambiguities in HLA-DRB1 generic typing were subjected to confirmatory typing using the PCR-single strand conformation polymorphism (SSCP) method. On the basis of these results, an 'Interpretation Program for Koreans'was developed for the 45 probe Dynal DRB test. RESULTS: Among 3,000 Koreans tested by the 36 probe Dynal DRB test, 456 cases (15.2%) showed ambiguities. In 95% of the ambiguity cases (433/456) and 99.2% of the total cases tested (433/3,000), the'most probable type'could be expected from the DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans and these results were in accordance with the confirmatory typing results as well as the results given by the 'Interpretation Program for Koreans'. Similarly, the 'Most Probable'could be assigned by the program in 99.4% (348/350) of the cases tested with the 45 probe Dynal DRB test. CONCLUSIONS: Ambiguity in the Dynal DRB test was observed in >15% of the Korean samples tested. The majority (95%) of the ambiguities could be resolved on the basis of HLA-DRB1 gene frequencies and DRB1-B3/B4/B5 associations in Koreans. Furthermore, using the program developed in this study, the correct assignment of DRB1 generic types was possible without additional typing in the majority (>99%) of the cases tested.
Dichlororibofuranosylbenzimidazole ; DNA ; Gene Frequency ; HLA-DR Antigens* ; HLA-DRB1 Chains

PURPOSE: It was suggested that immunogenic region of E7 proteins of human papillo- mavirus (HPV) type 16 encompass casein kinase (CK) II phosphorylation site and the resulting negative charge may affect the various biologic function of E7 protein. This study was undertaken to analyze the change of antigenic characteristics of HPV type 16, E7 oncoprotein according to phosphorylation. MATERIALS AND METHODS: We produced two monoclonal antibodies (VD6 and IB10) which showed different reactivities to E7 proteins expressed from bacteria or extracted from CaSki cell. These reaction were analyzed by Western blotting. Also the antigenic sites estimation of these antibodies using nested deletion sets was done. On the basis of above experiments, we performed in vitro phosphorylation assay using CK II and its specific inhibitor, DRB (5, 6-dichloro-l-beta-D-ribofuranosylbenzimidazole), to analyze the IB10 reactivity to E7 oncoproteins according to phosphorylation. RESULTS: In Westem blot analysis, VD6 and IB10 antibodies reacted strongly to bacterially expressed E7 protein. But using E7 extracted from CaSki cell, VD6 reacted to 2.0 kDa E7 protein whereas IB10 showed weak reactivity. The antigenic sites estimation of these antibodies showed that antigenic site of VD6 was located in amino terminal region and that of IB10 in the middle portion in the range of approximate amino acid 25-45. The antigenic site of IB10 might contain the possible phosphorylation sites (Ser-31, 32) in E7. Considering this, the different reactivities of IB10 to E7 proteins expressed in bacteria and extracted from CaSki cell might be due to phosphorylation. In in vitro phosphorylation assay using CK II, the phosphorylation of E7 increased according to reaction time. And this phosphorylation reduced the reactivity of IB10 to E7 protein whereas the reactivity of VD6 did not change. Also the reactivity of IB10 to E7 protein increased in a dose dependent manner with CK II specific inhibitor, DRB treated CaSki cell extracts. CONCLUSION: These result showed the antigenecity is affected by the degree of phosphorylation of E7 protein.
Antibodies ; Antibodies, Monoclonal ; Bacteria ; Blotting, Western ; Casein Kinases ; Cell Extracts ; Dichlororibofuranosylbenzimidazole ; Human papillomavirus 16 ; Humans* ; Oncogene Proteins ; Phosphorylation* ; Reaction Time

BACKGROUND AND OBJECTIVES: The prognosis and natural history of bradycardia related to drugs such as beta-blockers and non-dihydropyridine calcium channel blockers are not well known. SUBJECTS AND METHODS: We retrospectively analyzed 38 consecutive patients (age 69+/-11, 21 women) with drug-related bradycardia (DRB) between March 2005 and September 2007. A drug-associated etiology for the bradycardia was established based on the medical history and patient response to drug discontinuation. The mean follow-up duration was 18+/-8 months. RESULTS: The initial electrocardiogram (ECG) showed sinus bradycardia (heart rate < or =40/min) in 13 patients, sinus bradycardia with junctional escape beats in 18 patients, and third-degree atrioventricular (AV) block in seven patients. Drug discontinuation was followed by resolution of bradycardia in 60% of patients (n=23). Among them, five (17.8%) patients resumed taking the culprit medication after discharge and none developed bradycardia again. Bradycardia persisted in 10 (26.3%) patients despite drug withdrawal, and a permanent pacemaker was implanted in seven of them. Third-degree AV block, QRS width, and bradycardia requiring temporary transvenous pacing were significantly associated with the bradycardia caused by drugs. CONCLUSION: Beta-blockers were the most common drugs associated with DRB. However, in one quarter of the cases the DRB was not associated with drugs; in these patients permanent pacemaker implantation should be considered.
Arrhythmias, Cardiac ; Atrioventricular Block ; Bradycardia ; Calcium Channel Blockers ; Dichlororibofuranosylbenzimidazole ; Electrocardiography ; Follow-Up Studies ; Humans ; Natural History ; Prognosis ; Retrospective Studies ; United Nations

AIMTo study the direct effect and kinetics of sodium quercetin-7,4'-disulphate (SQDS) on recombinant human protein kinase CK2 holoenzyme.

METHODSThe recombinant human CK2 holoenzyme activity was assayed by detecting incorporation of 32P of [gamma-32P] ATP into the substrate in various conditions.

RESULTSThe recombinant human CK2 was a second messenger (Ca2+, cAMP and cGMP) independent protein kinase. The characterization and function of the reconstituted holoenzyme were consistent with those of native CK2. SQDS was shown to strongly inhibit the holoenzyme activity of recombinant human protein kinase CK2 with an IC50 of 4.4 mumol.L-1, which was more effective than DRB and A3, known CK2 special inhibitors. Kinetic studies of SQDS on recombinant human CK2 showed: the inhibition was competitive with ATP and noncompetitive with casein.

CONCLUSIONSQDS is a potent inhibitor of protein kinase CK2. This study provide experimental basis for the development of more effective inhibitors of CK2 and for clinical application of SQDS in the future.

Casein Kinase II ; Dichlororibofuranosylbenzimidazole ; pharmacology ; Enzyme Inhibitors ; pharmacology ; Humans ; Kinetics ; Protein-Serine-Threonine Kinases ; antagonists & inhibitors ; metabolism ; Quercetin ; analogs & derivatives ; pharmacology ; Recombinant Proteins ; antagonists & inhibitors ; metabolism

OBJECTIVETo investigate the effects of transcriptional inhibitors 5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole (DRB) and alpha-Amanitin on the localization of Nrf2 in the nucleus.

METHODSA549 cells were treated with DRB (50 mg/L) or alpha-Amanitin (2.5 mg/L)for 1 h and 6 h in serum-free medium, respectively. The expressions of Nrf2, HO-1, NQO1 and AKR1C were detected by Western blotting analysis. The localization of Nrf2 was determined by laser scanning confocal microscopy after cells were treated with either DRB or agr:-Amanitin for 1 h.

RESULTSThe expressions of Nrf2 and Nrf2-ARE gene batteries HO-1, AKR1C and NQO1 were decreased after 6 h treated with either DRB or alpha-Amanitin. The expression of SC35 was up-regulated but RNA Pol II was down-regulated; Y12 and NPC did not significantly change. The localization of Nrf2 in the cell nucleus did not change significantly.

CONCLUSIONDRB and alpha-Amanitin can down-regulate the expression of Nrf2 and its targeting proteins HO-1, AKR1C and NQO1, but may have no effect on the localization of Nrf2.

20-Hydroxysteroid Dehydrogenases ; genetics ; metabolism ; Alpha-Amanitin ; pharmacology ; Carcinoma, Non-Small-Cell Lung ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Dichlororibofuranosylbenzimidazole ; pharmacology ; Heme Oxygenase-1 ; genetics ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; pathology ; NF-E2-Related Factor 2 ; genetics ; metabolism ; Nucleic Acid Synthesis Inhibitors ; pharmacology