Functional polymorphism of the human arylamine N-acetyltransferase type 1 gene caused by (CT)-T-190 and G(560)A mutations

Human N-acetyltransferase type 1 (NAT1) catalyses the N- or O-acetylation of various arylamine and heterocyclic amine substrates and is able to bioactivate several known carcinogens. Despite wide inter-individual variability in activity, historically, NAT1 was considered to be monomorphic in nature. However, recent reports of allelic variation at the NAT1 locus suggest that it may be a polymorphically expressed enzyme. In the present study, peripheral blood mononuclear cell NAT1 activity in 85 individuals was found to be bimodally distributed with approximately 8% of the population being slow acetylators. Subsequent sequencing of the individuals having slow acetylator status showed all to have either a (CT)-T-190 or G(560)A base substitution located in the protein encoding region of the NAT1 gene. The (CT)-T-190 base substitution changed a highly conserved Arg(64), which others have shown to be essential for fully functional NAT1 protein. The (CT)-T-190 mutation has not been reported previously and we have named it NAT1*17. The G(560)A mutation is associated with the base substitutions previously observed in the NAT1*10 allele and this variant (NAT1*14) encodes for a protein with reduced acetylation capacity. A novel method using linear PCR and dideoxy terminators was developed for the detection of NAT1*14 and NAT1*17. Neither of these variants was found in the rapid acetylator population. We conclude that both the (CT)-T-190 (NAT1*17) and G(560)A (NAT1*14) NAT1 structural variants are involved in a distinct NAT1 polymorphism. Because NAT1 can bioactivate several carcinogens, this polymorphism may have implications for cancer risk in individual subjects. (C) 1998 Chapman & Hall Ltd.

Association between chronic fatigue syndrome and the corticosteroid-binding globulin gene ALA SER224 polymorphism

Chronic fatigue syndrome (CFS) is characterized by idiopathic fatigue of greater than 6 months' duration with postexertional exacerbation and many other symptoms. A trend toward relative hypocortisolism is described in CFS. Twin and family studies indicate a substantial genetic etiologic component to CFS. Recently, severe corticosteroid-binding globulin (CBG) gene mutations have been associated with CFS in isolated kindreds. Human leukocyte elastase, an enzyme important in CBG catabolism at inflammatory sites, is reported to be elevated in CFS. We hypothesized that CBG gene polymorphisms may act as a genetic risk factor for CFS. A total of 248 patients with CFS defined by Centers for Disease Control criteria, and 248 controls were recruited. Sequencing and restriction enzyme testing of the CBG gene coding region allowed detection of severe CBG gene mutations and a common exon 3 polymorphism (c.825G --> T, Ala-Ser(224)). Plasma CBG levels were measured in 125 CFS patients and 198 controls by radioimmunoassay. Total and free (calculated and measured) cortisol levels were ascertained in single samples between 8-10 a.m. The age of onset (mid 30s) and gender ratio (2.2:1, female:male) of the patients were similar to those reported in U.S. epidemiologic studies. A trend toward a preponderance of serine(224) homozygosity among the CFS patients was noted, compared with controls (chi(2) = 5.31, P = 0.07). Immunoreactive-CBG (IR-CBG) levels were higher in Serine/Alanine (Ser/Ala) than Ala/Ala subjects and higher again in Ser/Ser subjects, this effect was strongest in controls; Ser/Ser: 46.1 +/- 1.8 (n = 31, P = 0.03) vs. Ser/Ala: 42.4 +/- 1.0 (n = 56, P = 0.05) vs. Ala/Ala: 40.8 +/- 1.7 mug/mL (n = 21). Despite higher CBG levels, there was a nonsignificant trend toward lower total and free plasma cortisol in serine allele positive patients, total cortisol: Ser/Ser: 13.3 +/- 1.4 (n = 34) vs. Ser/Ala: 14.0 +/- 0.7 (n = 66) vs. Ala/Ala: 15.4 +/- 1.0 (n = 23). Homozygosity for the serine allele of the CBG gene may predispose to CFS, perhaps due to an effect on hypothalamic-pituitary-adrenal axis function related to altered CBG-cortisol transport function or immune-cortisol interactions.

Activation of calcineurin in human failing heart ventricle by endothelin-1, angiotensin II and urotensin II

1 The calcineurin (CaN) enzyme-transcriptional pathway is critically involved in hypertrophy of heart muscle in some animal models. Currently there is no information concerning the regulation of CaN activation by endogenous agonists in human heart. 2 Human right ventricular trabeculae from explanted human ( 14 male/2 female) failing hearts were set up in a tissue bath and electrically paced at 1Hz and incubated with or without 100 nM endothelin-1 (ET-1), 10 mu M, angiotensin-II (Ang II) or 20 nM human urotensin-II (hUII) for 30 min. Tissues from four patients were incubated with 200 nM tacrolimus (FK506) for 30 min and then incubated in the presence or absence of ET-1 for a further 30 min. 3 ET-1 increased contractile force in all 13 patients (P < 0.001). Ang II and hUII increased contractile force in three out of eight and four out of 10 patients but overall nonsignificantly (P > 0.1). FK506 had no effect on contractile force (P = 0.12). 4 ET-1, Ang II and hUII increased calcineurin activity by 32, 71 and 15%, respectively, while FK506 reduced activity by 34%. ET-1 in the presence of FK506 did not restore calcineurin activity (P = 0.1). 5 There was no relationship between basal CaN activity and expression levels in the right ventricle. Increased levels of free phosphate were detected in ventricular homogenates that were incubated with PKC epsilon compared to samples incubated without PKCe. 6 Endogenous cardiostimulants which activate G alpha q-coupled receptors increase the activity of calcineurin in human heart following acute (30 min) exposure. PKC may contribute to this effect by increasing levels of phosphorylated calcineurin substrate.

Allelic Variation Investigation of the Estrogen Receptor Within an Australian Multiple Sclerosis Population

Multiple Sclerosis (MS) is a central nervous system (CNS) chronic inflammatory demyelinating disease leading to various neurological disabilities. The disorder is more prevalent for women with a ratio of 3:2 female to male. Objectives: To investigate variation within the estrogen receptor 1 (ESR1) polymorphism gene in an Australian MS case-control population using two intragenic restriction fragment length polymorphisms; the G594A located in exon 8 detected with the BtgI restriction enzyme and T938C located in intron 1, detected with PvuII. One hundred and ten Australian MS patients were studied, with patients classified clinically as Relapsing Remitting MS (RR-MS), Secondary Progressive MS (SP-MS) or Primary Progressive MS (PP-MS). Also, 110 age, sex and ethnicity matched controls were investigated as a comparative group. No significant difference in the allelic distribution frequency was found between the case and control groups for the ESR1 PvuII (P = 0.50) and Btg1 (P = 0.45) marker. Our results do not support a role for these two ESR1 markers in multiple sclerosis susceptibility, however other markers within ESR1 should not be excluded for potential involvement in the disorder.

No Association Between MTHFR A1298C and MTRR A66G Polymorphisms, and MS in an Australian Cohort

Multiple sclerosis (MS) is a complex neurological disease that affects the central nervous system (CNS) resulting in debilitating neuropathology. Pathogenesis is primarily defined by CNS inflammation and demyelination of nerve axons. Methionine synthase reductase (MTRR) is an enzyme that catalyzes the remethylation of homocysteine (Hcy) to methionine via cobalamin and folate dependant reactions. Cobalamin acts as an intermediate methyl carrier between methylenetetrahydrofolate reductase (MTHFR) and Hcy. MTRR plays a critical role in maintaining cobalamin in an active form and is consequently an important determinant of total plasma Hcy (pHcy) concentrations. Elevated intracellular pHcy levels have been suggested to play a role in CNS dysfunction, neurodegenerative, and cerebrovascular diseases. Our investigation entailed the genotyping of a cohort of 140 cases and matched controls for MTRR and MTHFR, by restriction length polymorphism (RFLP) techniques. Two polymorphisms: MTRR A66G and MTHFR A1298C were investigated in an Australian age and gender matched case-control study. No significant allelic frequency difference was observed between cases and controls at the α = 0.05 level (MTRR χ^2 = 0.005, P = 0.95, MTHFR χ^2 = 1.15, P = 0.28). Our preliminary findings suggest no association between the MTRR A66G and MTHFR A1298C polymorphisms and MS.

Enzyme deactivation in fixed bed reactors with michaelis-menten kinetics

Rate expression for enzyme poisoning which are consistent with a Michaelis-Menten main reaction are used to analyze the performance of a fixed bed reactor containing immobilized enzyme. When enzyme deactivation results from the irreversible bonding of a product molecule to an existing substrate-enzyme complex, it is shown that minimum enzyme activity can occur in the interior of the bed, well away from the ends. This suggests that bed sectioning techniques may enable direct evaluation of fundamental poisoning mechanisms.

Enzyme immobilization in porous solid supports - penetration of immobilized enzyme

The process of enzyme immobilization under the diffusion-controlled regime (i.e., fast attachment of enzyme compared to its diffusion) is modeled and theoretically solved in this article. Simple and compact solutions for the penetration depth of immobilized enzyme and the bulk enzyme concentration versus time are presented. Furthermore, the conditions for the validity of our solutions are also given in this article so that researchers can discover when the theoretical solutions can be applied to their systems.

Inactivation of human arylamine N-acetyltransferase 1 by the hydroxylamine of p-aminobenzoic acid

Human N-acetyltransferase 1 (NAT1) is a widely distributed enzyme that catalyses the acetylation of arylamine and hydrazine drugs as well as several known carcinogens, and so its levels in the body may have toxicological importance with regard to drug toxicity and cancer risk. Recently, we showed that p-aminobenzoic acid (PABA) was able to down-regulate human NAT1 in cultured cells, but the exact mechanism by which PABA acts remains unclear. In the present study, we investigated the possibility that PABA-induced down-regulation involves its metabolism to N-OH-PABA, since N-OH-AAF functions as an irreversible inhibitor of hamster and rat NAT1. We show here that N-OH-PABA irreversibly inactivates human NAT1 both in cultured cells and cell cytosols in a time- and concentration-dependent manner. Maximal inactivation in cultured cells occurred within 4 hr of treatment, with a concentration of 30 muM reducing activity by 60 +/- 7%. Dialysis studies showed that inactivation was irreversible, and cofactor (acetyl coenzyme A) but not substrate (PABA) completely protected against inactivation, indicating involvement of the cofactor-binding site. In agreement with these data, kinetic studies revealed a 4-fold increase in cofactor K-m, but no change in substrate K-m for N-OH-PABA-treated cytosols compared to control. We conclude that N-OH-PABA decreases NAT1 activity by a direct interaction with the enzyme and appears to be a result of covalent modification at the cofactor-binding site. This is in contrast to our findings for PABA, which appears to reduce NAT1 activity by down-regulating the enzyme, leading to a decrease in NAT1 protein content. BIOCHEM PHARMACOL 60;12: 1829-1836, 2000. (C) 2000 Elsevier Science Inc.

MHCWeb: Converting a WWW database into a knowledge-based collaborative environment

The World Wide Web (WWW) is useful for distributing scientific data. Most existing web data resources organize their information either in structured flat files or relational databases with basic retrieval capabilities. For databases with one or a few simple relations, these approaches are successful, but they can be cumbersome when there is a data model involving multiple relations between complex data. We believe that knowledge-based resources offer a solution in these cases. Knowledge bases have explicit declarations of the concepts in the domain, along with the relations between them. They are usually organized hierarchically, and provide a global data model with a controlled vocabulary, We have created the OWEB architecture for building online scientific data resources using knowledge bases. OWEB provides a shell for structuring data, providing secure and shared access, and creating computational modules for processing and displaying data. In this paper, we describe the translation of the online immunological database MHCPEP into an OWEB system called MHCWeb. This effort involved building a conceptual model for the data, creating a controlled terminology for the legal values for different types of data, and then translating the original data into the new structure. The 0 WEB environment allows for flexible access to the data by both users and computer programs.

Cyanogenic polymorphism in Eucalyptus polyanthemos Schauer subsp vestita L. Johnson and K. Hill (Myrtaceae)

Plant cyanogenesis, the release of cyanide from endogenous cyanide-containing compounds, is an effective herbivore deterrent. This paper characterises cyanogenesis in the Australian tree Eucalyptus polyanthemos Schauer subsp. vestita L. Johnson and K. Hill for the first time. The cyanogenic glucoside prunasin ((R)-mandelonitrile beta-D-glucoside) was determined to be the only cyanogenic compound in E. polyanthemos foliage. Two natural populations of E. polyanthernos showed quantitative variation in foliar prumasin concentration, varying from zero (i.e. acyanogenic) to 2.07 mg CN g(-1) dry weight in one population and from 0.17 to 1.98 mg CN g(-1) dry weight in the other. No significant difference was detected between the populations with respect to the mean prunasin concentration or the degree of variation in foliar prunasin, despite significant differences in foliar nitrogen. Variation between individuals was also observed with respect to the capacity of foliage to catabolise prunasin to form cyanide. Moreover, variation in this capacity generally correlated with the amount of prunasin in the tissue, suggesting genetic linkage between prunasin and beta-glucosidase. (C) 2002 Elsevier Science Ltd. All rights reserved.