Study of Exchange Phenomenon of Mycoplasma pneumoniae in Children from 1997-2002 / 소아과

PURPOSE: Mycoplasma pneumoniae(M. pneumoniae) is classified into two groups(I and II) by difference of DNA sequences in P1 protein. Between these two groups, there are some different immune responses and disease severity. M. pneumoniae pneumonia have epidemic outbreaks occurring every three to seven years and these outbreaks are related with rising of either group I or II. We studied cases of M. pneumoniae pneumonia during the past six years(November 1996-October 2002), to evaluate the prevalence and yearly distribution of each group. METHODS: We enrolled 504 patients out of 547 patients, who were admitted to the Department of Pediatrics, Sung-Ae and Kwangmyung Sung-Ae General Hospital from November 1996 to October 2002. They were diagnosed as M. pneumoniae pneumonia by clinical characteristics and indirect particle agglutination test of M. pneumoniae. To classify into two groups, the group specific polymerase chain reaction amplification were performed using specific oligonucleotide primers designed for P1 gene genotyping. RESULTS: Group I(91.7%) occured more frequently than group II(8.3%) during the study period. There were outbreaks of M. pneumoniae pneumonia in 1997 and 2000, which showed epidemics of M. pneumoniae pneumonia were occuring every three or four years, but there was no exchange phenomenon between the two groups. CONCLUSION: Group I was more prevalent than group II with a three years cycle of epidemic outbreak from 1997 to 2002 in Korea. But, six years of research is a relatively short time to compare immune responses, disease severity and exchange phenomenon between the two groups. Further follow-up study will be needed for the epidemiologic and clinical studies of M. pneumoniae in Korea.

Pancreatic Islets Transplantation through Spleenic Pulp and Peritoneum Using Hollow Fiber in Streptozotocin-induced Diabetic Nude Mouse / 대한소아내분비학회지

PURPOSE: Recently islets transplantation has been become a hot issue in insulin dependant diabetes mellitus. This study is aimed to review the technical method of islet isolation, pruification, and microencapsulation in animal model and to study the actual ability of transplated islets on controlling hyperglycemia. Finally, we want to know whether hollow fiber model for immunoislation in islet transplantation is effective in diabetic nude mouse. Method: We use 5-6 weeks old Spregue-Dawley rats as donor. After midline incision, collagenase was infused to proximal common bile duct and pancreas was extracted. With HBSS treatment and discontinuous density gradient centrifugation, islets were isolated. From 500-1,000 numbers of islets were transplanted to 6 strepozotocin-induced nude mice via upper pole of spleen and serial blood glucose level of nude mice were checked from conjunctival veins. Also, we examined transplanted-islets in spleen histologically with light and electron microscopy. Finally, after impregnation of 500 to 1,000 numbers of islets to 2-4 hollow fibers(Amicon R, H1P30-43 type, M.W:30,000) for immunoisolation, we inserted hollow fiber into peritoneum of 3 streptozotocin-induced nude mice and checked blood glucose level serially. Results: 1) Isolation of islets from rats was done successfully and we could calculate the number of islets under microscopy. 2) In 3 diabetic nude mice without islet transplantation(control group), all of them revealed hyperglycemia above 200mg/dL after 5days from strptozotocin injection. After then, blood glucose level was ranged from 300 to 500mg/dL persistently and all of them were died after 120 days. 3) We could observe the transplabted-islets in spleen with microscopy. Blood glucose level began to be controlled after 5 days from transplantation in 3 diabetic nude mice and all of 6 diabetic nude mouses revealed normoglycemia after 25 days from transplantation. 4) Islets transplantation with holler fibers into peritoneum of diabetic nude mice was not satisfactory. Although no technical difficulty was occured, persistent hyperglycemia was observed and all of 3 diabetic nude mice were died. CONCLUSION: Though islet transplantation through spleen was sucessful in diabetic nude mouse, further study is needed to clear the cause of failure of hollow fiber model in islets transplantation.

p53 Mutation and Functional Analyses by Using Yeast Functional Assay / 대한암학회지

PURPOSE: Mutation of the p53 tumor suppressor gene is the most common genetic defect in all human tumors. Because of the widespread mutations and polymorphism in the p53 gene, the conventional screening methods cannot distinguish between polymorphisms or functionally silent mutations and inactivating mutations. It is well known that plasmids can be generated by homologous recombination in vivo in the yeast by cotransforming the PCR product with a linearized yeast expression vector encoding part of a gene and a selectable marker gene. The aim of this study is to develop more easy and reliable method for functional assay of p53 mutation. MATERIALS AND METHODS: We constructed a gap vector which can reliably and conveniently be used to screen p53 mutations in a simple yeast growth assay. The gap vector was constructed as follows: About 100 bp DNA fragments containing parts of N- and C- terminal portion of p53 were cloned into XbaI/SmaI and HindIII/XhoI sites of yeast expressing vector, respectively. The gap vector was obtained by double cutting with SmaI and HindIII followed by gel elution. Yeast was transformed with the reporter vector containing three tandem copies of the consensus p53 binding site by lithium acetate-mediated method. RT-PCR amplification of p53 transcripts from cell lines or tumor tissues was carried out. To investigate whether p53 gene is mutated or not, yeast containing reporter gene was cotransformed with PCR product and linearized gap vector, plated on SD medium minus histidine, and incubated for 3 days. The colonies on selective media were isolated and characterized. RESULTS: The tumor tissues examined were one hepatocellular carcinoma, three breast cancers, two stomach cancers and two colon cancers. One hepatocellular carcinoma tissue had mutation in both alleles of the p53 gene, and 7 cancer tissues had heterozygous mutations in the p53 gene. The result of functional assay was well correlated with mutational analysis by sequencing. CONCLUSION: p53 functional assay system might be easy and reliable method for functional screening of p53 on tumor tissues and this might be used for screening of other mutated gene. This technique, FASAY, requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.