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Delayed hypersensitivity to 5-fluoro uracil associated with reduced dihydropyrimidine dehydrogenase deficiency
POSTER ABSTRACTS decreased level by blockade of neuronal activity with tetrodotoxin. An excitation dependent release might suggest a regulatory role of these compounds. This hypothesis is particularly interesting in view of recently cloned specific UTP receptors.
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F A L C I P A R U M HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE AS A CHEMOTHERAPEUTIC TARGET: COMPARISON OF THE HUMAN AND PARASITE ENZYMES
PLASMODIUM
Keough, D.T. 1'2, Ng, A J, Emmerson, B.T. 2 and de Jersey, j 1 Departments of Biochemistry I and Medicine 2, University of Queensland, Brisbane, Australia 4072
43
DELAYED HYPERSENSITIVITY TO 5-FLUORO URACIL A S S O C I A T E D WITH REDUCED DIHYDROPYRIMIDINE DEHYDROGENASE DEFICIENCY _Duley J.A. and *Nethersell A.B.W. Purine Research Laboratory, UMDS Guy's Hospital, London, and *Clinical Oncology, North Devon District Hospital, Barnstaple, UK
Sensitivity to 5-fluorouracil (5FU) therapy has previously been shown to be associated with heterozygosity for inherited deficiency of dihydropyrimidine dehydrogenase (DPD). Objectives We studied a UK patient who showed a delayed reaction to 5FU, with near fatal results. DPD activity was assessed as a possible explanation of his profound reaction. Design and Methods A 69 year old man with a metastatic adenocarcinoma of gastrointestinal origin received 1.2 g 5FU by bolus injection. He experienced mild discomfort in his mouth and slight diarrhoea, but 5 days post-injection his blood count was only slightly depressed. Nine days following the first injection, a second bolus was administered. The patient had a mild febrile reaction, but six days later was seen with profound mucositis, extensive rash and exfoliation, and severely depressed white cell, neutrophil and platelet counts. He was admitted but nearly died of neutropenic sepsis. A malignant lymph node in his left neck completely regressed. Results The white cell DPD of the patient was assayed and found to be 2.5 pmol/h/mg protein, which was approximately half the control mean. Conclusion This patient appeared to show a delayed hypersensitivity to 5FU, with serious side-effects, as a result of reduced DPD activity. The level of activity was consistent with heterozygosity for inherited DPD deficiency, although a full family study has not been undertaken to confirm this.
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THE MANAGEMENT OF GOUT Bryan Emmerson, Department of Medicine, Princess Alexandra Hospital, Brisbane, Q, 4102, Australia
Objectives The relative merits of the various therapies for acute gout will be discussed, including treatment with colchicine, nonsteroidal antiinflammatory drugs and corticosteroids. In addition, methods of prophylaxis against acute attacks and their effectiveness will be considered, Methods Gout can be prevented by correcting the underlying hyperuricaemia. One way of achieving this is to determine the cause of the hyperuricaemia in the individual patient and correct it. Correctable causes include obesity, hypertension, regular alcohol consumption, high purine intake, inadequate urine volume, or the consumption of drugs such as diuretics which elevate urate concentrations. Where these do not apply or cannot be corrected, therapy with urate-lowering drugs, either uricosurics or allopurinol, should correct the hyperuricaemia. Conclusion In 90% of patients with gout, effective management should make acute gout a thing of the past. In the remaining 10%, challenging problems remain which will be considered. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
Objectives Plasmodium falciparum is the most serious of the parasites that cause malaria. Unlike humans, protozoan parasites are unable to synthesize purine mononucleotides de nero 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 SO 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) Mel. Biochem. Parasitol. 30:123-134
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SUBSTRATE S P E C I F I C I T I E S AND SEQUENCE SIMILARITIES OF DEOXYNUCLEOSIDE KINASES; IMPLICATIONS FOR CHEMOTHERAPY S. Eriksson and L. Wang, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, S-75321 Uppsala, Sweden
Objectives Deoxynucleoside kinases are key enzymes in the activation of several anti cancer and anti viral drugs. The are four kinases in animal cells; cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). All these enzymes have now been purified and characterised and the sequences for their coding regions determined. We will summarise some of these results and their implications for chemotherapy. Design and Methods The cDNA:s for dGK and TK2 were cloned, sequenced and expressed by using RT-PCR methods based on amino acid sequence information from the purified proteins. The enzymatic properties of purified recombinant TK2 and dGK were determined. Results Recombinat TK2 and dGK showed very similar substrate kinetics to those of the purified "native" enzymes. The mitochondrial kinases show 40% homology to human dCK cDNA but only 12% to the TK1 cDNA. Conclusions Two distinct enzyme families among the cellular deoxynucleoside kinases can be identified; the dCK-herpes virus TK family and the TK1 family with important evolutionary and functional consequences. 253
45
F A L C I P A R U M HYPOXANTHINEGUANINE PHOSPHORIBOSYLTRANSFERASE AS A CHEMOTHERAPEUTIC TARGET: COMPARISON OF THE HUMAN AND PARASITE ENZYMES
PLASMODIUM
Keough, D.T. 1'2, Ng, A J, Emmerson, B.T. 2 and de Jersey, j 1 Departments of Biochemistry I and Medicine 2, University of Queensland, Brisbane, Australia 4072
43
DELAYED HYPERSENSITIVITY TO 5-FLUORO URACIL A S S O C I A T E D WITH REDUCED DIHYDROPYRIMIDINE DEHYDROGENASE DEFICIENCY _Duley J.A. and *Nethersell A.B.W. Purine Research Laboratory, UMDS Guy's Hospital, London, and *Clinical Oncology, North Devon District Hospital, Barnstaple, UK
Sensitivity to 5-fluorouracil (5FU) therapy has previously been shown to be associated with heterozygosity for inherited deficiency of dihydropyrimidine dehydrogenase (DPD). Objectives We studied a UK patient who showed a delayed reaction to 5FU, with near fatal results. DPD activity was assessed as a possible explanation of his profound reaction. Design and Methods A 69 year old man with a metastatic adenocarcinoma of gastrointestinal origin received 1.2 g 5FU by bolus injection. He experienced mild discomfort in his mouth and slight diarrhoea, but 5 days post-injection his blood count was only slightly depressed. Nine days following the first injection, a second bolus was administered. The patient had a mild febrile reaction, but six days later was seen with profound mucositis, extensive rash and exfoliation, and severely depressed white cell, neutrophil and platelet counts. He was admitted but nearly died of neutropenic sepsis. A malignant lymph node in his left neck completely regressed. Results The white cell DPD of the patient was assayed and found to be 2.5 pmol/h/mg protein, which was approximately half the control mean. Conclusion This patient appeared to show a delayed hypersensitivity to 5FU, with serious side-effects, as a result of reduced DPD activity. The level of activity was consistent with heterozygosity for inherited DPD deficiency, although a full family study has not been undertaken to confirm this.
44
THE MANAGEMENT OF GOUT Bryan Emmerson, Department of Medicine, Princess Alexandra Hospital, Brisbane, Q, 4102, Australia
Objectives The relative merits of the various therapies for acute gout will be discussed, including treatment with colchicine, nonsteroidal antiinflammatory drugs and corticosteroids. In addition, methods of prophylaxis against acute attacks and their effectiveness will be considered, Methods Gout can be prevented by correcting the underlying hyperuricaemia. One way of achieving this is to determine the cause of the hyperuricaemia in the individual patient and correct it. Correctable causes include obesity, hypertension, regular alcohol consumption, high purine intake, inadequate urine volume, or the consumption of drugs such as diuretics which elevate urate concentrations. Where these do not apply or cannot be corrected, therapy with urate-lowering drugs, either uricosurics or allopurinol, should correct the hyperuricaemia. Conclusion In 90% of patients with gout, effective management should make acute gout a thing of the past. In the remaining 10%, challenging problems remain which will be considered. CLINICAL BIOCHEMISTRY, VOLUME 30, APRIL 1997
Objectives Plasmodium falciparum is the most serious of the parasites that cause malaria. Unlike humans, protozoan parasites are unable to synthesize purine mononucleotides de nero 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 SO 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) Mel. Biochem. Parasitol. 30:123-134
46
SUBSTRATE S P E C I F I C I T I E S AND SEQUENCE SIMILARITIES OF DEOXYNUCLEOSIDE KINASES; IMPLICATIONS FOR CHEMOTHERAPY S. Eriksson and L. Wang, Department of Veterinary Medical Chemistry, Swedish University of Agricultural Sciences, The Biomedical Centre, Box 575, S-75321 Uppsala, Sweden
Objectives Deoxynucleoside kinases are key enzymes in the activation of several anti cancer and anti viral drugs. The are four kinases in animal cells; cytosolic thymidine kinase (TK1) and deoxycytidine kinase (dCK) and the mitochondrial thymidine kinase (TK2) and deoxyguanosine kinase (dGK). All these enzymes have now been purified and characterised and the sequences for their coding regions determined. We will summarise some of these results and their implications for chemotherapy. Design and Methods The cDNA:s for dGK and TK2 were cloned, sequenced and expressed by using RT-PCR methods based on amino acid sequence information from the purified proteins. The enzymatic properties of purified recombinant TK2 and dGK were determined. Results Recombinat TK2 and dGK showed very similar substrate kinetics to those of the purified "native" enzymes. The mitochondrial kinases show 40% homology to human dCK cDNA but only 12% to the TK1 cDNA. Conclusions Two distinct enzyme families among the cellular deoxynucleoside kinases can be identified; the dCK-herpes virus TK family and the TK1 family with important evolutionary and functional consequences. 253