In view of the potential of replicase protein as a diagnostic reagent for human caliciviruses (HuCVs), we have cloned and over-expressed this gene from the Snow Mountain-like Korean strains in Escherichia coli as a fusion protein with glutathione S-transferase (GST, and described the preliminary antigenic characterization of the recombinant products. Each 470bp fragment corresponding to highly conserved region of RNA-dependent RNA polymerase was generated by RT-PCR from stools of two diarrheal children, cloned in pMOSBlue T-vector, and subcloned between the EcoRI and SalI restriction sites of pGEX-47-3, a GST gene fusion vector, yielding pGCVpol. This construct expressed a Snow Mountain-like HuCV replicate under the control of the IPTG-inducible pac promoter. An extract prepared by sonication of the E. coli cell inclusion bodies bearing pGCVpol products was purified and analyzed by SDS-PAGE. After Coomassie blue staining, it was shown that the recombinant replicase migrated on the gels with an approximate molecular mass of 46.5 kDa, that was subsequently cleaved into a 26 kDa GST fragment and a 20.5 kDa replicase protein upon digestion with thrombin protease. The replicase was recognized on immunoblotting with the sera from symptomatic children with the HuCV-associated diarrhea but not by asymptomatic sera from adults. The results presented the first biological activity of individually expressed HuCV replicase subunit and provided important reagents for diagnosis of HuCV infection.
Adult ; Child ; Clone Cells ; Diagnosis ; Diarrhea ; Digestion ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; Gels ; Gene Fusion ; Glutathione Transferase ; Humans ; Immunoblotting ; Inclusion Bodies ; Indicators and Reagents ; RNA Replicase ; Snow* ; Sonication ; Staphylococcal Protein A ; Thrombin

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8Flla/68/US, with exceptions of HuCV185/87/Korea and HuCV1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV185/87/Korea and HuCV1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.
Amino Acid Motifs ; Amino Acid Sequence ; Animals ; Child ; Clone Cells ; DNA, Complementary ; Epidemiology ; Gastroenteritis ; Genetic Variation ; Genome ; Genotype ; Humans* ; Korea* ; Norwalk virus ; Polymerase Chain Reaction ; RNA Replicase ; Snow

Human caliciviruses (HuCVs) cause sporadic cases and outbreaks of acute gastroenteritis (AGE). Three maior genogrovps of HuCVs have been described including the Norwalk virus (NV)-, the Snow Mountain virus (SMA)-, and the Sapporo-genogroups. This study describes the detection and genetic variation of HuCVs from hospitalized infants with AGE in Korea by RT-PCR and sequencing. The cDNA fragments of 206 to 470bp corresponding to the region of 3 primer pairs (36/35, 35/51 or 3/51) in the polymerase region of NV were generated. Of 185 stools screened, 8% were positive by RT-PCR and their sequences showed that all strains contained the GLPSG and YGDD motifs which are conserved for HuCVs. Amino acid (aa) sequence analysis showed that these strains can be divided into 3 maior genogroups. High conservation was observed in that one strain shares 100% of as sequence with Southampton virus, another shares 99% with the Sapporo virus, and six strains share 90 to 95% with Snow Mountain virus. However, significant sequence variation was also found in other strains. This study indicates that all maior genogroups of HuCVs are circulating in Korea.
Disease Outbreaks ; DNA, Complementary ; Gastroenteritis ; Genetic Variation ; Genotype ; Humans* ; Infant ; Korea* ; Norwalk virus ; RNA Replicase* ; Sapovirus ; Sequence Analysis ; Snow

With improvement of anterior fixation devices, anterior instrumentation-fusion in treatment of thoracolumbar fracture becomes as effective as posterior interbody fusion which has mainly been used up to now. Also, computerized tomography enables us to diagnose the spinal fracture accurately including retropulsive bony fragment and degree of narrowing of spinal canal. We analyzed 18 cases which were treated with anterior interbody fixation in thoracolumbar fracture from Mar. 1977 to Oct. 1986 in Orthopaedic dept. of National Medical Center. The results were as follows ; 1. The mechanism of injury involved falling down in 7 cases, traffic accident 5 cases, and miner injury in 4 cases. 2. The clsssification of fracture by Denis method was compression fracture in 4 cases, bursting fracture in 11 cases, Seat-belt injury in 1 case, Fx-dislocation in 2 cases. 3. The methods of internal fixation were iliac bone graft only in 8 cases, anterior Harrington rod in 2 cases, Webb implant in 6 cases, and Zielke rod in 2 cases. 4. The average preoperative kyphotic angulation was 19.5(range 10 to 22) and immediate postoperative angulation was 10.1(range 2–16). The correction rate was 48%, and the loss of correction was 2.8(range 2–13). The final correction rate was 38.1% with implants, 29.1% without implants. 5. Early operation can get better neurologic recovery. 6. Firm and stsble fixation device is needed.
Accidental Falls ; Accidents, Traffic ; Fractures, Compression ; Methods ; Miners ; Spinal Canal ; Spinal Fractures ; Transplants

Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative a analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and PRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-P-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETA5-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Clone Cells* ; Cloning, Organism* ; DNA, Complementary ; Electrophoresis ; Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Humans ; Plasmids ; Polymerase Chain Reaction ; RNA, Viral

No abstract available.
Baculoviridae* ; Bombyx* ; Coinfection*

The ischemic coitis proximal to obstructive colon cancer was firstly reported by MacCallum and Kaisch in 1947. Nowsdays, its clinical importance and the study of pathogenesis is reported frequently. Because of the rare inciedence of this complicated condition this lesion could be overlooked which then could result anastomotic leakage, fecal fistula and peritonitis. Recently, we have examined a 61 year-old Korean male patient who had the complaints of acute abdominal pain and bloody diarrhea. The diagnosis of gangrenous ischemic colitis associated with obstucting rectal cancer was confirmed on the basis of sigmoidoscopic and operative and associated histopathological findings. After prompt surgical intervention, the patient was discharged with good improvement. The literatures are reviewed here with the case presentation.
Abdominal Pain ; Anastomotic Leak ; Colitis, Ischemic* ; Colonic Neoplasms ; Diagnosis ; Diarrhea ; Fistula ; Humans ; Male ; Middle Aged ; Peritonitis ; Rectal Neoplasms

For the purpose of prenatal diagnosis of CAH, genetic linkage analysis by HLA genotyping with lymphocytes and cultured amniotic cells were performed in a family at risk in which two consecutive children had been affected with SW CAH. In addition, the response of serum 17-OHP to intravenous ACTH was determined in obligate carrier parents, and 17-OHP concentration of amniotic fluid was also measured at 16 weeks of gestation. As might be expected, the baseline levels of 17-OHP in obligate parents were significantly higher than that of normal control. Although the post stimulation response of 17-OHP to ACTH in the mother (I-2) was significantly higher than that of normal control, the post stimulation levels of 17-OHP were in normal range in the father (I-1). The 17-OHP level (5.7 ng/ml) in the amniotic fluid showed intermediate value compared to Pang's report (normal less than 30 ng/ml, CAH greater than 12.0 ng/ml) suggesting heterozygote of the fetus. Genetic linkage analysis by HLA genotyping with cultured amniotic cells revealed heterozygote in their fetus (II-3) who has received one chromosome No,6 containing HLA haplotype A24, B40, Cw3 (normal allele for 21-OH) from the father and the other chromosome No,6 containing HLA haplotype A2, Bw62, Cw4 (mutant allele for 21-OH D) from the mother. In conclusion, attempts to detect heterozygote for 21-OH deficiency by ACTH stimulation test were partially successful and prenatal diagnosis of CAH by the hormone studies in ammiotic fluid requires reliable values in normal, heterozygotes and patients group, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
*Adrenal Hyperplasia, Congenital/*diagnosis/enzymology/genetics ; Adult ; Amniocentesis ; Cells, Cultured ; Female ; Fetal Diseases/*diagnosis/enzymology ; HLA Antigens/analysis ; Heterozygote Detection ; Humans ; Pedigree ; Pregnancy ; *Prenatal Diagnosis ; Steroid Hydroxylases

Hemodynamics in 60 normal, 15 simple goiter and 40 hyperthyroid subjects were determined from non-invasive Wezler's method. Hemodynamic changes in hyperthyroid patients were compared with those of normal subjects. The relationships of the cardiac output, aging,131I uptake rate and PEP/LVET to the hemodynamic changes in Hyperthyroid patients were also discussed. 1) The hemodynamics of normal subjects were same as those of simple goiter. 2) The heart rate and cardiac output were increased very significantly in hyperthyroid patients but they appeared to decrease with age. The stroke volume was increased slightly in the hyperthyroid patients, but remained unlateerd even with aging. Peripheral arterial resistance in hyperthyroid patients was decreased but appeared to increase with age. 3) As the131I uptake rate increased, the heart rate, cardiac output also increased, but PEP/LVET as an index of myocardial contractility and the peripheral arterial resistance decreased. 4) PEP/LVET as an index of myocardial contractility in hyperthyroid patients was markedly reduced. 5) The relationship of stroke volume and the rate of the heart to cardiac output in normal subjects were reasonably linear, and peripheral arterial resistance has negative relation to cardiac output. However the heart rate, though increased in most patients with thyrotoxicosis, bears no close relations to the level of cardiac output increment. The relationshop of stroke volume to cardiac output in hyperthyroid patients is reasonably linear and peripheral arterial resistance has negative correlation with the cardiac output.
Aging ; Cardiac Output ; Goiter ; Heart ; Heart Rate ; Hemodynamics* ; Humans ; Stroke Volume ; Thyrotoxicosis

PURPOSE: This study has been planned to generate a replication-competent adenovirus which replicates in a cancer cell-specific manner, thus minimizing the side effects and toxicity of cancer gene therapy. MATERIALS AND METHODS: we have generated an E1B 19 kD attenuated recombinant adenoviruses, Ad-TERT-delta19 and Ad-mTERT-delta19, which encode E1A gene driven by the wild type hTERT and modified m-hTERT promoter containing additional c-myc and Sp1 binding sites in the backbone of Ad-deltaE1B19. The in vitro efficacy and specificity of the hTERT and m-hTERT promoter have been evaluated by the comparison of viral replication and cytopathic effect in cancer cells and normal cell lines. To assess anti-tumor effect and safety of hTERT or m-hTERT promoter driven replication competent adenoviruses, tumor regression after subcutaneous injection in subcutaneous C33A xenografts and lacZ expression after systemic injection in organs were examined. RESULTS: The activation of hTERT or m-hTERT promoter was significantly up-regulated only in hTERT-positive cells, but not in hTERT-negative cells. Moreover, the activity of m-hTERT promoter was substantially increased in hTERT-positive cancer cells, but not in hTERT-negative cells. While Ad-TERT-delta19 replicated in and induced cytopathic effect in cancer and in some normal cell lines, Ad-mTERT-delta19 enhanced viral replication and cytopathic effect in cancer cells only. Furthermore, the growth of established human cervical carcinoma in nude mice was significantly suppressed by intratumoral injection of Ad-mTERT-delta19. CONCLUSIONS: The use of m-hTERT promoter is not only useful in the regulation of therapeutic gene expression but also that replication-competent oncolytic adenovirus under the control of m-hTERT promoter may be a new promising tool for the treatment of human malignancies.
Adenoviridae* ; Animals ; Binding Sites ; Cell Line ; Gene Expression ; Genes, Neoplasm ; Heterografts ; Humans* ; Injections, Subcutaneous ; Mice ; Mice, Nude ; Sensitivity and Specificity ; Telomerase*