| Project/Area Number |
02454175
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
細菌学
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| Research Institution | The Institute of Medical Science, The University of Tokyo |
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Principal Investigator |
NAKAMURA Yoshikazu Institute of Medical Science, Associate, 医科学研究所, 助教授 (40114590)
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| Co-Investigator(Kenkyū-buntansha) |
EGAWA Kohji Institute of Medical Science, Professor, 医科学研究所, 教授 (00012724)
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| Project Period (FY) |
1990 – 1991
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| Project Status |
Completed (Fiscal Year 1991)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 1991: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 1990: ¥4,800,000 (Direct Cost: ¥4,800,000)
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| Keywords | Pneumocystis carinii / pneumonia / AIDS / opportunistic infection / polymerase chain reaction / DNA diagnosis / 5S ribosomal RNA / surface antigen / モノクロ-ナル抗体 / 遺伝子クロ-ニング / 予防・治療 |
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| Research Abstract |
Pneumocystis carinii is an opportunistic pathogen which often causes fatal pneumonia in patients under immunosuppressed or immune deficient conditions due to AIDS, cancer chemotherapy or immunosuppressive therapy for organ transplantation. P. carinii is a eukaryotic microorganism and infects many mammalian hosts. Continuous in vitro culture of P. carinii has not been established and thereby the difficulty of mass cultivation impedes progress in the study of this organism. Nonetheless, it is necessary to pursue molecular approaches to P. carinii studies.
1. Surface Glycoproteins. The major surface immunodeterminants of P. carinii is a group of proteins called P115. They are composed of six major isoelectric variants and highly glycosylated with mannose. The frequent generation of anti-Pll5 monoclonal antibodies implicates a strong antigenic activity of these molecules. Deglycosylation analysis revealed that the sugar moiety of P115 plays a crucial role in the antigenic activity.
2. PI 15
… More
cDNA Cloning. P. carinii cDNA library was constructed using lambdagtll vector. The library was screened with antibodies against the P115 protein moiety, and 40 positive clones were isolated. Nucleotide sequence analyses revealed that they were classified to at least three similar but not identical groups. They share the same epitope site to monoclonal antibodies. Furthermore, rabbit polyclonal antibodies raised against one of these cDNA-lacZ fusion proteins reacted with the intact P115 molecules. Therefore, we were convinced of our cloning of P115 cDNA. The observed heterogeneity in the cDNA structure might reflect heterogeneity in P. carinii organisms or variability of the surface structure as found in other parasitic protozoa.
3. Diagnosis with Polymerase Chain Reaction. We have successfully utilized the polymerase chain reaction (PCR) method to detect P. carinii organisms by amplifying the 5S RDNA sequence in various clinical and animal samples. The detection was highly sensitive and specific. Extensive trials manifested efficacy of the PCR method in sputum samples from patients who suffered from pneumonia. The PCR-positive patients were then administered daily with pentamidine or sulfamethoxazole/trimethoprim and were monitored by the PCR assay. These trials demonstrated the efficacy of applying PCR to the monitoring of therapy to P. carinii pneumonia. _ Less
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