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Plasmodium falciparum hypoxanthine-guanine phosphoribosyltransferase as a chemotherapeutic target: Comparison of the human and parasite enzymes
POSTER ABSTRACTS Results ATP content was decreased after 8 hours of incubation in (3) (14.47 +- 0.95*) while no change was observed in (4) (16.71 +1.23) or (2) (17.98 ± 1.42) vs (1) (16.37 ± 1.42). In (5) and (6) ATP was enhanced (18.39 _+ 1.29" and 21.49 _+ 1.92"). Adenosine increased in (3) (0.43 ± 0.05*), (4) (0.41 +_ 0.07*), (5) (1.25 -+ 0.25*) and (6) (0.99 +- 0.18") vs control (0.25 -+ 0.05) but not with (2) (0.26 +_ 0.07). Conclusion DIPY decreases ATP concentration in isolated myocytes by mechanisms other than NTI but increases adenosine. A/R + DIPY protected ATP and increased adenosine production.
first, were proved to contain more constituents with the use of X-ray diffractometer. Conclusion Analysis with a micro area X-ray diffractometer could distinguish precise structures of crystalline materials in the body.
THE ORIGIN OF THE MOST COMMON MUTATION OF A D E N I N E PHOSPHORIBOSYLTRANSFERASE DEFICIENCY AMONG J A P A N E S E GOES BACK TO A PREHISTORIC ERA Naoyuki Kamatani, Chihiro Terai, Seong Yoon Kim 1, Ching-Lang Chen 2, Hisashi Yamanaka, Masayuki Hakoda, Shin Totokawa and Sadao Kashiwazaki, Institute of Rheumatology, Tokyo Women's Medical College, KS BLDG, 9-12 Wakamatsu-cho, Shinjuku-ku, Tokyo 162, Japan; 1Reumatism Center, Hanyang University Hospital, Seoul, Korea; 2Special Clinic of Gout, Taipei Municipal Ho-Ping Hospital, Taipei, Taiwan
ADA deficiency in humans results in impaired lymphoid development and combined immunodeficiency disease. Objective To understand the metabolic basis for the lymphoid phenotype associated with ADA deficiency. Design and Methods Genetically engineer ADA-deficient mice that retain critical features associated with ADA deficiency in humans. Results ADA-deficient mice were produced by a two stage genetic engineering strategy that restored ADA to the placentas of ADA-deficient fetuses. This prevented the serious purine metabolic disturbances and fetal liver damage that would otherwise occur. The resulting mice completely lacked ADA when born and were characterized by high levels of circulating adenosine and deoxyadenosine, and pronounced disturbances in deoxynucleotide metabolism. The ADA-deficient mice were severely lymphopenic and had rudimentary spleens and thymuses. Conclusion We have produced ADA-deficient mice that retain critical features associated with ADA deficiency in humans. These mice provide an unprecedented opportunity to study the lymphoid-specific metabolic consequences of ADA deficiency and to explore various therapeutic strategies for treating this and related metabolic disorders.
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Objectives To search for the origin of the most common mutation (APRT*J) accounting for 68% of the disease-causing genes of adenine phosphoribosyltransferase deficiency among Japanese. Design and Methods Geographical distribution of the mutant genes found in the homozygotes in J a p a n was studied, and the incidences of A P R T * J gene among Japanese, Koreans and Taiwanese were determined. Results The A P R T * J mutation is distributed nearly uniformly in the four main islands of Japan and Okinawa, suggesting a very early origin. The incidences of the heterozygotes with A P R T * J gene among the general populations of Japan, Korea and Taiwan were 0.73% (7/955), 0.53% (2/356) and 0% (0/231), respectively. Conclusion Since the A P R T * J mutation is present in Koreans and Okinawans who share ancestors only before the Yayoi era (3rd century BC-3rd century AD), the origin of the A P R T * J mutation predates 300 BC.
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MICROANALYSIS OF PATHOLOGICAL CRYSTALS A N D URINARY C A L C U L I Kaneko K 1, Fujimori S", Kamatani N ~, Yamanaka H 3 and Akaoka 12, 1Central Laboratory of Analytical Biochemistry, 2Department of Internal Medicine, Teikyo University School of Medicine; 3Institute of Rheumatology, Tokyo Women's Medical College; Tokyo, Japan
Objectives Pathological crystals are known to cause arthritis. Urinary calculi are often found in patients with gout or hypouriceamia. Microanalysis was carried out to determine fine structures of such small materials as pathological crystals and urinary calculi. Methods Crystalline materials from patients with osteoarthritis, gout or hypouriceamia, were examined. For analysis, a micro area X-ray diffractometer and an infrared spectrophotometer (IR) were used. Results Calcium deposits gave similar X-ray spectra, which agreed well with hydroxyapatite, in every area analyzed. IR spectra additionally indicated carbonate groups in the deposits. Monosodium urate crystals in synovial fluid, which could not been examined with IR, were analyzed precisely with X-ray diffractometer. Urinary calculi, which were analyzed with IR CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
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MAKING MICE MORE LIKE PEOPLE: THE CASE OF ADENOSINE DEAMINASE ( A D A ) DEFICIENCY M.R. Blackburn, S.K. Datta and R.E. Kellems, Department of Biochemistry, Baylor College of Medicine, Houston, Texas
P L A S M O D I U M F A L C I P A R U M HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE A S A CHEMOTHERAPEUTIC TARGET: COMPARISON OF T H E H U M A N A N D PARASITE ENZYMES Keough, D.T. 1'2, Ng, A. 1, Emmerson, B.T. 2 and de Jersey, j.1, Departments of Biochemistry' and Medicine 2, University of Queensland, Brisbane, Australia 4072
Objectives Plasmodium falciparum is the most serious of the parasites that cause malaria. Unlike humans, protozoan parasites are unable to synthesize purine mononucleotides de novo and rely on the availability of preformed purine bases from their host cell. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a key enzyme in the salvage of these bases, making it a potential target for chemotherapy. Design and Methods In order to obtain sufficient quantities of human and parasite HPRT, the cDNA coding for each of these two enzymes was cloned into the pT7-7 expression vector and the enzymes expressed in E. coli S(5 606 cells. Results Both the human and parasite HPRT enzymes have been purified to homogeneity in mg quantities. Other workers (Queen et al., 1988) have found the parasite HPRT to be very unstable and this has been one of the major obstacles in studying this enzyme. This problem has now been overcome. Conclusions Studies of the properties of the human and parasite HPRTs have highlighted differences between the active sites of these two enzymes. This is the first step in the design ofinhibitors as potential chemotherapeutic agents. Queen, S.A., Jagt, D.V. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30: 123-134. 263
first, were proved to contain more constituents with the use of X-ray diffractometer. Conclusion Analysis with a micro area X-ray diffractometer could distinguish precise structures of crystalline materials in the body.
THE ORIGIN OF THE MOST COMMON MUTATION OF A D E N I N E PHOSPHORIBOSYLTRANSFERASE DEFICIENCY AMONG J A P A N E S E GOES BACK TO A PREHISTORIC ERA Naoyuki Kamatani, Chihiro Terai, Seong Yoon Kim 1, Ching-Lang Chen 2, Hisashi Yamanaka, Masayuki Hakoda, Shin Totokawa and Sadao Kashiwazaki, Institute of Rheumatology, Tokyo Women's Medical College, KS BLDG, 9-12 Wakamatsu-cho, Shinjuku-ku, Tokyo 162, Japan; 1Reumatism Center, Hanyang University Hospital, Seoul, Korea; 2Special Clinic of Gout, Taipei Municipal Ho-Ping Hospital, Taipei, Taiwan
ADA deficiency in humans results in impaired lymphoid development and combined immunodeficiency disease. Objective To understand the metabolic basis for the lymphoid phenotype associated with ADA deficiency. Design and Methods Genetically engineer ADA-deficient mice that retain critical features associated with ADA deficiency in humans. Results ADA-deficient mice were produced by a two stage genetic engineering strategy that restored ADA to the placentas of ADA-deficient fetuses. This prevented the serious purine metabolic disturbances and fetal liver damage that would otherwise occur. The resulting mice completely lacked ADA when born and were characterized by high levels of circulating adenosine and deoxyadenosine, and pronounced disturbances in deoxynucleotide metabolism. The ADA-deficient mice were severely lymphopenic and had rudimentary spleens and thymuses. Conclusion We have produced ADA-deficient mice that retain critical features associated with ADA deficiency in humans. These mice provide an unprecedented opportunity to study the lymphoid-specific metabolic consequences of ADA deficiency and to explore various therapeutic strategies for treating this and related metabolic disorders.
83
Objectives To search for the origin of the most common mutation (APRT*J) accounting for 68% of the disease-causing genes of adenine phosphoribosyltransferase deficiency among Japanese. Design and Methods Geographical distribution of the mutant genes found in the homozygotes in J a p a n was studied, and the incidences of A P R T * J gene among Japanese, Koreans and Taiwanese were determined. Results The A P R T * J mutation is distributed nearly uniformly in the four main islands of Japan and Okinawa, suggesting a very early origin. The incidences of the heterozygotes with A P R T * J gene among the general populations of Japan, Korea and Taiwan were 0.73% (7/955), 0.53% (2/356) and 0% (0/231), respectively. Conclusion Since the A P R T * J mutation is present in Koreans and Okinawans who share ancestors only before the Yayoi era (3rd century BC-3rd century AD), the origin of the A P R T * J mutation predates 300 BC.
84
MICROANALYSIS OF PATHOLOGICAL CRYSTALS A N D URINARY C A L C U L I Kaneko K 1, Fujimori S", Kamatani N ~, Yamanaka H 3 and Akaoka 12, 1Central Laboratory of Analytical Biochemistry, 2Department of Internal Medicine, Teikyo University School of Medicine; 3Institute of Rheumatology, Tokyo Women's Medical College; Tokyo, Japan
Objectives Pathological crystals are known to cause arthritis. Urinary calculi are often found in patients with gout or hypouriceamia. Microanalysis was carried out to determine fine structures of such small materials as pathological crystals and urinary calculi. Methods Crystalline materials from patients with osteoarthritis, gout or hypouriceamia, were examined. For analysis, a micro area X-ray diffractometer and an infrared spectrophotometer (IR) were used. Results Calcium deposits gave similar X-ray spectra, which agreed well with hydroxyapatite, in every area analyzed. IR spectra additionally indicated carbonate groups in the deposits. Monosodium urate crystals in synovial fluid, which could not been examined with IR, were analyzed precisely with X-ray diffractometer. Urinary calculi, which were analyzed with IR CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
85
86
MAKING MICE MORE LIKE PEOPLE: THE CASE OF ADENOSINE DEAMINASE ( A D A ) DEFICIENCY M.R. Blackburn, S.K. Datta and R.E. Kellems, Department of Biochemistry, Baylor College of Medicine, Houston, Texas
P L A S M O D I U M F A L C I P A R U M HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE A S A CHEMOTHERAPEUTIC TARGET: COMPARISON OF T H E H U M A N A N D PARASITE ENZYMES Keough, D.T. 1'2, Ng, A. 1, Emmerson, B.T. 2 and de Jersey, j.1, Departments of Biochemistry' and Medicine 2, University of Queensland, Brisbane, Australia 4072
Objectives Plasmodium falciparum is the most serious of the parasites that cause malaria. Unlike humans, protozoan parasites are unable to synthesize purine mononucleotides de novo and rely on the availability of preformed purine bases from their host cell. Hypoxanthine-guanine phosphoribosyltransferase (HPRT) is a key enzyme in the salvage of these bases, making it a potential target for chemotherapy. Design and Methods In order to obtain sufficient quantities of human and parasite HPRT, the cDNA coding for each of these two enzymes was cloned into the pT7-7 expression vector and the enzymes expressed in E. coli S(5 606 cells. Results Both the human and parasite HPRT enzymes have been purified to homogeneity in mg quantities. Other workers (Queen et al., 1988) have found the parasite HPRT to be very unstable and this has been one of the major obstacles in studying this enzyme. This problem has now been overcome. Conclusions Studies of the properties of the human and parasite HPRTs have highlighted differences between the active sites of these two enzymes. This is the first step in the design ofinhibitors as potential chemotherapeutic agents. Queen, S.A., Jagt, D.V. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30: 123-134. 263