BACKGROUND: Although phospholipase C (PLC)-B3 is thought to be a very important enzyme in intracellular signal transduction, the sophisticated and complicated purification steps make it difficult to obtain sufficient amount of protein to study regulation of its activity by G proteins or other proteins. In order to get large amount of PLC-B3 protein, I employed baculovirus expression system which is known to express large amount of functionally active proteins. METHODS: In order to make recombinant baculovirus which expresses PLC-B3 gene, partial cDNA of PLC-B3 which lacked 51 nucleotides was used to make full length PLC-B3 cDNA. By PCR, 5-end sequence of PLC-B3 was ligated into partial rat PLC-B3 cDNA and later cloned into pVL1393 transfer vector to make recombinant baculovirus. This recombinant baculovirus containing PLC-B3 sequence was used to infect Sf9 insect cells. RESULTS: Infection of Sf9 cells with recombinant baculovirus rendered expression of 152 kDa-PLC-B3 protein, which was confirmed by immunoblot assay and PLC activity assay. CONCLUSION: The whole length PLC-B3 cDNA was expressed in Sf9 cells using baculovirus expression system. By using it, homogeneous enzyme is expected to be purified to study precise activation and regulation mechanisms of PLC-B3.
Animals ; Baculoviridae* ; Clone Cells ; DNA, Complementary ; GTP-Binding Proteins ; Insects ; Nucleotides ; Phospholipases* ; Polymerase Chain Reaction ; Rats ; Sf9 Cells ; Signal Transduction ; Type C Phospholipases

No Abstract Available.
GTP-Binding Proteins* ; Receptors, Cell Surface*

No abstract available.
Spondylolisthesis*

BACKGROUND: The Chinese hamster ovary cells transfected with human TSH receptor cDNA (hTSHR-CHO), expressing functional human TSH receptors, are known to be more sensitive in detection of thyroid stimulating antibodies than FRTL-5 cells. There has been no report on the usefulness of these cells to measure thyroid stimulation blocking antibody (TSBAb) activity which is frequently found in patients with primary myxedema, METHODS: We established the optimal assay condition of measurement of TSBAb using hTSHR-CHO cells, and simultaneously measured TSBAb activities with FRTL-5 cells and with hTSHR-CHO cells in 49 patients with primary myxedema, compared them with their thyrotropin binding inhibitor immunoglobulin (TBII) activities. RESULTS: 1) hTSHR-CHO cells specifically bound bTSH and were stimulated by bTSH in terms of cyclic AMP generation in a dose dependent manner. 2) Myxedema IgG suppressed TSH-stimulated cAMP production of hTSHR-CHO cells in a dose dependent manner reaching plateau at the concentration of I g/L. Normal pooled IgG has no suppressive action at the concentration of less than 1 g/L, but caused significant suppression at the concentration of greater than 1g/L. 3) TSBAb activities measured by hTSHR-CHO cells in 49 patients with primary myxedema were as follows: Four of 25 TBII-negative cases (16%) and 22 of 24 TBII-positive cases (92%) had TSBAb activities. Most of TSBAb positive patients (95%), especially in TBII positive cases, showed very high activities of more than 90%. 4) TSBAb activities measured by hTSHR-CHO cells and those by FRTL-5 cells were both positive in 24 patients (49%), both negative in 18 patients (37%), and were discrepant in 7 patients (14%). The TSBAb activities measured with hTSHR-CHO cells and those measured with FRTL-5 cells were significantly correlated (r=0.71, p< 0.01). 5) Forty five percent of patients with primary myxedema had all of 3 kinds of activities (TBII, hTSHR-CHO cell TSBAb, FRTL-5 cell TSBAb), 37% of them had none of 3 activities and 18% of them had 1 or 2 kinds of activities only. CONCLUSION: The usefulness of hTSHR-CHO cells in measurements of TSBAb activities were confirmed. The TSBAb activities of most patients with primary myxedema measured by hTSHR-CHO cells were concordant with those measured by FRTL-5 cells. However, a small subset of patients (18%) had discrepant results in assays of TSH receptor antibodies according to the differences in TSH receptors (rat, human and porcine) used in assay. Such discrepancy may be explained by heterogeneity in epitopes for blocking TSH receptor antibodies.
Animals ; Antibodies ; Asian Continental Ancestry Group* ; Cricetinae ; Cricetulus* ; Cyclic AMP ; DNA, Complementary ; Epitopes ; Female ; Humans ; Humans* ; Hypothyroidism* ; Immunoglobulin G ; Immunoglobulins ; Immunoglobulins, Thyroid-Stimulating ; Myxedema ; Ovary* ; Population Characteristics ; Receptors, Thyrotropin* ; Thyroid Gland* ; Thyrotropin

BACKGROUND: Phospholipase C-beta4 (PLC-beta4) is known to be one of the most important signal transducing molecules; however, its biophysical and chemical characteristics are not well known due to the difficulty in purifying PLC-beta4 from bovine retina. In the present study, we used the baculovirus expression system in order to express and purify large amounts of PLC-beta4. With this system, we also tried to produce chimeric PLC-beta3/beta4 and PLC-beta4/beta3 protein in order to study the structure-activity relationship between N terminal and C terminal portion of PLC-betas. METHODS: I cloned PLC-beta4 to the baculovirus expression system by the polymerase chain reaction method and infected the PLC-beta4 to Sf9 cells. I purified recombinant PLC-beta4 proteins using sequential high performnance liquid chromatography (HPLC) by using the TSK phenyl-5PW column and the TSK heparin-5PW column. With this similar method, I was able to express chimeric PLC-beta3/beta4 and PLC-beta4/beta3 proteins. RESULTS: With the two step HPLC, I was able to purify PLC-beta4 by 30-fold; this purified PLC-beta4 contained PLC activity. I also expressed chimeric PLC-beta3/beta4 and PLC-beta4/beta3 using the baculovirus system, and their expression was confirmed by the immunoblot method. However, chimeric PLC-beta4/beta3 did not show PLC activity, while chimeric PLC-beta3/beta4 retained its PLC-activity. CONCLUSION: Expression of chimeric PLC-beta4 using the baculovirus system was an efficient method to obtain a large amount of protein. Moreover, this expression and purification method would be useful in studying the physical and chemical characteristics of this protein. In my study using chimeric PLC-beta protein by swapping the N terminal and C terminal portions of PLC-beta3 and beta4, chimeric protein lost its activity completely in PLC-beta4/beta3 chimera. This result suggested a minute change in the tertiary structure of the protein, which may significantly affect its function.
Baculoviridae ; Chimera ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Clone Cells ; Phospholipase C beta ; Phospholipases ; Polymerase Chain Reaction ; Proteins ; Retina ; Sf9 Cells ; Structure-Activity Relationship ; Type C Phospholipases

OBJECTIVE: The aim of our study was to compare the thickness and histopathologic changes in the fetal membrane between premature rupture of membranes (PROM) and intact membrane after delivery. METHODS: In a prospective study involving 31 patients who were divided into 4 groups such as <37 weeks without PROM, <37 weeks with PROM, >or=37 weeks without PROM, and >or=37 weeks with PROM, we measured the thickness of membrane and studied the histopathologic findings in vitro by light microscopy of histological sections. RESULTS: The membrane thickness of <37 weeks with PROM group was thinner (35.9 micrometer) than that (42.3 micrometer) of <37 weeks without PROM group, but there was no statistical significance. The membrane thickness of >or=37 weeks with PROM and >or=37 weeks without PROM were similar (25.6 micrometer, 26.0 micrometer). But the membrane thickness of >or=37 weeks with/without PROM was significantly thinner (25.8 micrometer) compared with that (38.9 micrometer) of <37 weeks with/without PROM. The histopathologic features of PROM positive group was amnionitis with neutrophilic infiltration, focally or diffusely necrotic change of amniotic membrane, separation of amniotic membrane and degeneration of chorionic villi. CONCLUSION: The thickness of fetal membrane between PROM group and intact membrane group was not different but the thickness of fetal membrane between <37 weeks and >or=37 weeks was statistically significant. The histopathologic change of PROM positive group was prominent as amnionitis. Further evaluation will be needed about the relationship between membrane thickness and PROM.
Amnion ; Chorioamnionitis ; Chorionic Villi ; Extraembryonic Membranes ; Female ; Humans ; Membranes* ; Microscopy ; Neutrophils ; Pregnancy ; Prospective Studies ; Rupture*

No abstract available.
Thyroid Gland*

BACKGROUND: An accurate and rapid method for specise identification of coagulase negative staphylococci(CNS) has been increasingly necessary for the clinical significance and planning the management of patients with staphylococcal infections. Recently, it has been reported that there is a highly conserved area on their 60KDa heat shock protein(HSP60) gene sequences between the interspecies of CNS and it can be amplified by a set of universal degenerate primer. This led us our attention to focus on whether the PCR-based RFLP method using Mse / restriction enzyme could be a useful tool for the species identification of CNS. METHODS: In the present study, we performed PCR-based RFLP analysis using a set of degenerate primers covering HSP60 and Mse / restriction enzyme on the reference strains and 25 clinical isolates(10 of S. epidermidis, 10 of S. haemolyticus, 4 of S. lugdunensis and 1 of S. warneri) which were previously identified by the API-STAPH, Vitek GPI card and/or with conventional biochemical test. RESULT: All the seven reference strains revealed that each strain has a distinct electrophoresed band patterns with combination of different number (up to 8) and size of fragments. And these distinct band patterns showed remarkable concordance with the seven reference strains and 25 clinical isolates. CONCLUSION: These result strongly suggest that the PCR-RFLP method using degenerate primers covering the HSP60 gene and Mse / digestion enzyme offer a convenient and accurate tool for species-specific identification of CNS.
Chaperonin 60* ; Coagulase* ; Digestion ; Heat-Shock Proteins* ; Hot Temperature* ; Humans ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Shock ; Staphylococcal Infections

No abstract available.
Chorion* ; Chorionic Villi*

A study was performed to investigate a relationship between the status of T lymphocyte subsets in the peripheral blood and the pathogenesis of erythema nodosum leprosum (ENL) reactions. T lymphocyte subsets, B lymphocytes and serum immnnoglobulin levels were explored in 40 leprosy patients (10 tuberculoid (TT), 17 lepromatous (LL), and 13 lepromatous with ENL). In patients with LL, suppressor cells increased and helper cell diminished, resulting in a decrease in the helper/suppressor (Th/Ts) ratio to 0.86 + 0.20, as compared with controls (1. 77 + 0.41). But in ENL patients, there was a significant diminution of suppressor cells and an increase of helper cells, resulting in an increase in Th/Ts ratio to 2.08 + 0.30. Four weeks after treatment of ENL, Th/Ts ratio decreased to l.18+ 0.31 again. The mean percentages of B cells and serum immunoglobulin levels in with LL. and ENL increased significantly compared with controls. Only IgG increased in patients with ENL compared with patients with LL. The results in patiients with TT did not differ significantly from those of controls. The findings suggest that cell-mediated immune responses, imbalance in T lymphocyte subsets, may play a role in the pathogenesis of ENL, either directly or by inducing an antibody critical to the formation of the immune complex
Antigen-Antibody Complex ; B-Lymphocytes ; Erythema Nodosum* ; Erythema* ; Humans ; Immunoglobulin G ; Immunoglobulins ; Leprosy ; T-Lymphocyte Subsets* ; T-Lymphocytes, Helper-Inducer