We investigated whether the two variant elements of CRE(TGcCGTCA[5'CRE], TGACcTCA[3'CRE]) in the 5'flanking region of the rat TRH gene, which are different from the CRE consensus sequence(5'-TGACGTCA-3') by one base pair, are responsive to cAMP, and whether the one base pair difference is responsible for the degree of cAMP responsiveness of the gene. When CA 77 cells were stimulated with forskolin and isobutylmethylxanthine for 4 hours, the level of TRH mRNA was increased by only two fold. The transient gene expression study using serial 5'deletion of the TRH gene in PC12 cells showed that the region between-113 and-77, which includes 5'CRE, was crucial for the cAMP resonsiveness. When the plasmid, which contains the 30 bp oligonucleotide including either 5'CRE or 3'CRE ligated to the enhancerless RSV promoter, was transfected into PC12 cells, it did not significantly affect not only the basal transcription but cAMP responsiveness. The 65 bp oligonucleotide including both 5'CRE and 3'CRE, however, increased both of the basal transcription and cAMP-stimulated transcription by 2-3 fold. When the sequence of 5'CRE was converted to that of the CRE consensus by replacing one base pair, the cAMP responsiveness was increased by two fold although the basal transcription was not increased. The one base pair mutant of 3'CRE increased both of the basal and cAMP-stimulated transcription by 3-4 fold. These results suggest that there are the two variant CREs in rat TRH gene, which are relatively weak CRE compared to the CREs of other neuropeptide genes and cooperative for the activation of both the basal and cAMP-stimulated transcription. The one base pair difference of the variant CREs from the CRE consensus sequence is responsible for the weak responsiveness to cAMP.
Animals ; Base Pairing ; Colforsin ; Consensus ; Consensus Sequence ; Cyclic AMP ; Gene Expression ; Neuropeptides ; PC12 Cells ; Plasmids ; Rats ; Response Elements ; RNA, Messenger ; Thyrotropin ; Thyrotropin-Releasing Hormone

BACKGROUND: We previously demonstrated that a GRE/TRE composite sequence, which is located between 200 bp and 220 bp relative to the transcriptional start site of rat TRH gene, is responsible for the dexamethasone (DEX)- and TPA-induced transcriptional activation, and the transcriptional activation by DEX is mediated by interaction between glucocorticoid receptor (GR) and a TRE-binding transcriptional factor such as c-Jun. However, a non-specific binding with the transciption factors can not be excluded as the mutants used in the previous report could not inhibit the binding of GR and c-Jun completely, and it remains unclear which one of the two TRE-like sequences is critical for the interaction of the two transcription factors. METHODS: Luciferase expressing plasmids that contain a part of rat TRH promoter including the composite GRE sequence or its mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX or/and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX and TPA increased the transcriptional activity of the wild type composite sequence by 3 folds and 4 folds, respectively, and the combined stimulation increased the activity by 10 folds. The mutants of which all 6 nucleotides of the GRE half site were replaced and removed almost did not bind to GR and eould not enhance the transcriptional activity at all in response to DEX. The GRE-deleted mutant bound to c-Jun with a remarkably lower affinity and showed a lower response to TPA, whereas the GRE-replaced mutant bound to c-Jun with a similar affinity and showed a similar response to TPA compared to those of the wild type. In response to the combined simulation with DEX and TPA, the mutants showed 30-40% of the trancriptional activity of the wild type. Basal transcriptional activity of all the TRE mutants was significantly lower than that of the wild type. While they almost could not bind to c-Jun, their binding affinity to GR was comparable to that of the wild type. Whereas the DEX- and TPA-induced transcriptional activity of 5 TRE mutant was 10% and 15% of that of the wild type, it responded to those agents in a similar pattern as the wild type. The 3 TRE mutant and the mutant of both TRE sites did not respond to DEX and TPA. The GRE-deleted mutant hardly formed the DNA-protein complex as did the wild type, while the GRE -replaced mutant could form the complex in a less amount with nuclear extract of HeLa celL CONCLUSION: These results suggest that GRE/TRE composite sequence of rat TRH gene specifically binds to GR and c-Jun, providing a site for interaction between the two transcription factors, and that both TRE sites play an important role in basal transcription, and that the 3 TRE site is more critical in the interaction between GRE and TRE for DEX-induced transcriptional activation. (J Kor Endocrinol 14:278-292, 1999)
Animals ; Dexamethasone ; Electrophoretic Mobility Shift Assay ; HeLa Cells ; Humans ; Luciferases ; Mutagenesis, Site-Directed* ; Nucleotides ; Plasmids ; Rats* ; Receptors, Glucocorticoid ; Response Elements* ; Transcription Factors ; Transcriptional Activation

BACKGROUND: We previously demonstrated that the promoter of rat TRH gene has GRE half site (TGTTCT) between -210 bp and -205 bp flanking with similar sequences of TPA response element (TRE), TAGTCA, at a distance of several base pairs from the GRE half site. It promps us to hypothesize that this composite GRE/TRE sequence can provide a site for interaction between glucocorticoid receptor (GR) and c-Jun. Thus, we investigated whether the composite sequence mediates transcriptional regulation induced by dexamethasone (DEX) and 12-O-tetradecanoyl phobol-13-acetate (TPA), and whether it binds GR and c-Jun. METHODS: A luciferase expressing plasmids that contain a part of rat TRH promoter including the composite sequence or their mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX increased the transcriptional activity of the plasmid containing the wild type GRE by 2.5 folds, and TPA increased the transcriptional activity by 4 folds. The simultaneous stimulation with DEX and TPA synergistically increased the transcriptional activity by 10 folds. Two mutants whose GRE half sits were altered showed no responses to DEX, and suppressed the TPA-induced or both agents-induced transcriptional activity by 50%. Two mutants whose TRE-like sites were altered suppressed the DEX-induced transcriptional activity by 20%, TPA-induced trarptional activity by 25%, and both agents-induced transcriptional activity by 50%. Gel retardation assay showed that the composite sequence fonned a complex with GR and its mutants bound to GR with remarkably less affinity. c-Jun also bound to the composite sequence to form two cornplexes with less affinity compared to the AP-1 consensus sequence. The mutants of the TRE-like sequence bound to c-Jun with a significantly lower affinity compared to that of the wild type. Simulateous binding of the composite sequence with GR and c-Jun did not form any larger complex. The complex of GR and the composite sequence was much smaller than that formed by c-Jun, suggesting that GR binds to the composite sequence as a monomer. CONCLUSION: These results suggest that the composite sequence of GRE half site and TRE-like site on the promoter of rat TRH gene provides binding sites for GR and c-Jun, which mediate the interaction between two signal transduction pathways. (J Kor Soc Endocrinol 14:265-277, 1999)
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid ; Animals ; Base Pairing ; Binding Sites ; Consensus Sequence ; Dexamethasone ; Electrophoretic Mobility Shift Assay ; HeLa Cells ; Humans ; Luciferases ; Plasmids ; Rats ; Receptors, Glucocorticoid ; Response Elements ; Signal Transduction ; Transcription Factor AP-1

No abstract available.
Bacteria*

BACKGROUND: Medtronic-Hall mechanical valve is a world widely using prosthesis. But, in Korea, the clinical result from Medtronic-Hall valve replacement is not frequenthy available. MATERIALS AND METHODS: From March 1986 to May 1990, 50 patients underwent valve replacement surgery with Medtronic-Hall valve at Pusan National University Hospital. Seventeen were male and thirty three were female and ra nging in age from 16 to 70 years of age (mean=35 years). RESULTS: The causes of valvular lesion were rheumatic in 43 patients, bicuspid aortic valve in 3 patients, degenerative lesion in three patients and bacterial endocarditis in one patient. The operative procedures were mitral valve replacement (MVR) in 38, aortic valve replacement (AVR) in 5 and double valve replacement (DVR) in 7. The most commonly used valve size was 21mm in AVR, 29mm in MVR. Concomitant surgical procedures were performed in 15 patients; left atrial thrombectomy in 9, left atrial auricle obliteration in 6 and tricuspid annuloplasty in 5 (Kay: 2, DeVega: 3). New York Heart Association functional class was mostly Class I or II (91.5%) preoperatively and Class IIIor IV (87.2%) after operation. The findings of postoperative echocardiogram of LAD, LVESD, LVEDD were reduced compared with preoperative period and ejection fraction was increased compared with preoperative period. Postoperative complications were massive bleeding in three, low cardiac output syndrome in two, thromboembolism in one and fulminant hepatitis in one patient. There were three hospital deaths and their causes were low cardiac output syndrome in two and rupture of left ventricle in one patient. The 5 year survival rate was 93.65+/-0.71% and 10 year actuarial survival rate was 88.27+/-6.42%. CONCLUSIONS: Medtronic-Hall mechanical valve has low valve related complication rate. It's durability and hemodynamic performance is comparable to other mechanical valves.
Aortic Valve ; Bicuspid ; Busan ; Cardiac Output, Low ; Endocarditis, Bacterial ; Female ; Heart ; Heart Valve Prosthesis ; Heart Ventricles ; Hemodynamics ; Hemorrhage ; Hepatitis ; Humans ; Korea ; Male ; Mitral Valve ; Postoperative Complications ; Preoperative Period ; Prostheses and Implants ; Rupture ; Surgical Procedures, Operative ; Survival Rate ; Thrombectomy ; Thromboembolism

No abstract available.
Actinomycosis*

BACKGROUND: The efficacy of the hemostasis of prophylactic aprotinin after cardiac valve replacement was evaluated from January 1994 to December 1996 at Pusan National University Hospital. MATERIAL AND METHOD: In a randomized study, 20 patients received aprotinin(2x106 KIU as a loading dose for 30 minutes after anesthesia, 1x106 KIU for priming and 5x105 KIU/hr as a maintenance dose from the completion of loading dose till skin closure) and another 20 untreated patients served as controls. RESULT: Aprotinin produced a significant reduction in postoperative blood loss compared with controls and significantly decreased total exposure to allogenic blood products compared with the control group(p<0.05). CONCLUSION: We conclude that aprotinin effectively reduces postoperative blood loss and trasfusion in patient undergoing cardiac valve replacement.
Anesthesia ; Aprotinin* ; Busan ; Heart Valves ; Heart* ; Hemostasis* ; Humans ; Postoperative Hemorrhage ; Skin ; Thoracic Surgery*

No abstract available.
Enterocolitis, Necrotizing*

OBJECTIVE: We investigated the expression of uPA and uPAR in eutopic endometrium of advanced stage endometriosis and control patients. METHODS: The 33 endometriosis patients and 32 controls were enrolled. Endometrial samples were obtained from 65 premenopausal women aged 29~44 years, undergoing laparoscopic surgery or hysterectomy for non-malignant lesions. Sufficient samples were collected from 33 patients with endometriosis stage III and IV and 32 controls without endometriosis confirmed by laparoscopic surgery. The mRNA expression of uPA and uPAR from eutopic endometrium were analyzed by RT-QC PCR. RESULTS: The mRNAs of uPA and uPAR were expressed in eutopic endometrium from endometriosis and normal controls throughout the menstrual cycle. Uterine endometrium from women with endometriosis expresses significantly (p<0.05) higher levels of u-PA mRNA than endometrium from normal women without endometriosis in the proliferative phase. There were no significant differences in expression of uPAR in eutopic endometrium between controls and endometriosis patients. CONCLUSION: These results suggest that eutopic endometrium from endometriosis patients may be more invasive and prone to peritoneal implantation because of greater u-PA mRNA expression than endometrium from women without endometriosis. Thus, increased proteolytic activity may be one etiology for the invasive properties of the endometrium resulting in the development of endometriosis.
Endometriosis* ; Endometrium* ; Female ; Humans ; Hysterectomy ; Laparoscopy ; Menstrual Cycle ; Polymerase Chain Reaction ; Proteolysis ; RNA, Messenger* ; Urokinase-Type Plasminogen Activator

No abstract available.
Krukenberg Tumor*