No description available.

No description available.

No description available.

A highly sensitive method, based on the use of a microscope fluorimeter and fluorogenic substrates, is being developed to measure rates of drug-metabolising enzyme reactions in small areas of cells in thin liver sections. The results described here illustrate a method in which reaction rates were measured in small groups of approximately 25 hepatocytes. The development of the method has been based initially on the measurement of the cytochrome. P-450-mediated ethoxyresorufin O-deethylase reaction in mouse hepatocytes. Fluorogenic substrates are known, however, for several other drug-metabolising enzymes and alternative fluorogenic substrates, namely pentoxyresorufin and benzyloxyresorufin, for different forms of P-450 have been used in this study. There has been significant improvement over an earlier method (Burke et al., 1983) of sectioning non-fixed, non-frozen pieces of liver. The left lobe of a mouse liver was stuck with cyanoacrylate glue to the Teflon stage of the Vibraslice cutting machine. The Teflon stage and attached liver lobe were immersed in a bath of Krebs-Henseleit physiological buffer. The specimen bath was moved towards the vibrating blade which cut through the liver so producing a section which was transferred with a fine brush to the surface of a slide of encapsulating resin on to which it was pinned. The section of liver was then superfused with the Krebs-Henseleit solution at 37 C on the heated stage of a microscope. The tip (10-12 um diameter) of a micropipette containing a solution of ethoxyresoruf in DMSO was manoeuvred onto the surface of the section using a micromanipulator. Selection of the cells to be measured and placement of the micropipette were carried out in transmitted light. The microscope was then switched to epi-illumination fluorescence mode. Ethoxyresoruf in was expelled from the micropipette on to the section by the application of a 2-5 second pulse of pressurised Nitrogen gas (approximately 10 lbf/in2). The volume of ethoxyresoruf in expelled was estimated as 0.405 0.027 ml using a combination of [3Hj-harmine and 1 mM ethoxyresorufin in the micropipette. The spread of substrate and the ensuing O-dealkylation reaction were confined to a circular area of the tissue approximately 100 um in diameter, comprising about 25 hepatocytes. During, the reaction, the fluorescence of the forming metabolite, resorufin, was measured continuously by means of a computerised photomultiplier system attached to the microscope. The measurement of fluorescence was from the surface layer of cells only. Since the method is at a stage in development, various, pieces of apparatus were subject to change. The excitation and emission filters were chosen so that the selected wavelengths of light were more appropriate for the metabolite resorufin than the substrate ethoxyresorufin. For excitation, a combination of a Vickers OG515 long wave pass filter (50% transmission between 370 nm and 560 nm), a Vickers TRITC exciter (50% transmission between 380 nm and 560 nm) and a Balzer TRITC dichroic mirror (10% transmission between 490 nm and 570 nm) were used. For emission, a Balzer TRITC dichroic mirror and a Barr and Stroud DL7S narrow band pass filter (maximum transmission of 49.2% at 590.1 nm) were used. The filters provided epi-illumination (incident) excitation light of 515-560 nm and selected emitted fluorescence of 580-600 run. A photomultiplier converted the photons of light into an electrical signal which was converted to a digital signal. Ideally the photomultiplier tube in use at each of the improvements was maximally sensitive to the emission wavelengths of resorufin. The digital signal was displayed either on a pen recorder or on a microcomputer, both of which resulted in a graph of relative fluorescence against time. From this graph three measurements were taken. These were: i) the initial reaction velocity (rate of linear rise of fluorescence intensity). ii) the highest fluorescence intensity attained, and iii) the time to reach the highest fluorescence intensity after the cessation of ethoxyresorufin application. The initial velocity had a closer correlation with the maximum reading (r = 0.75) than with the time at which the maximum occurred (r = 0.43). The results encompassed a wide range of values but did follow a normal distribution which meant that the Student's t-test could be used to test for significant differences between means. Means s.e. mean initial velocities measured in centrilobular and periportal regions of liver sections from control mice, were 2.7 0.32 and 1.9 0.28 fluor. unitsmin-1 respectively, suggesting no' difference between centrilobular and periportal regions. Pretreatment with MC increased these values to 55.0 + 3.5 and 49.9 3.3 respectively but again suggested no centrilobular/periportal difference, -Naphthoflavone produced less of an effect causing an increase from 11.2 1.7 to 52.9 10.6 in centrilobular regions and from 7.3 1.0 to 51.7 10.0 in periportal regions.

The metabolism of drugs and foreign compounds is a major influence on their pharmacological and toxicological effects. The haemoprotein cyt. P-450 exists as a multi-isozyme family which functions in the metabolism of a wide variety of foreign compounds. Biochemical information concerning the structure and catalytic function of cyt. P-450 isozymes from man is less extensive than from animal species. In this study several microsomal proteins including four cyt. P-450 isozymes, NADPH-cyt. P-450 reductase, epoxide hydrolase and other unidentified proteins were purified from adult human liver; the methodologies previously employed to isolate cyt. P-450 isozymes were improved and now allow better resolution and recovery of human cyt. P-450. Comparison of the physical and chromatographic properties of the cyt. P-450 isozymes isolated (cyt. P-450 7:3 cyt. P-4507:4 and cyt. P-4507:5) indicated that they were very similar and probably identical. N-terminal amino acid sequence and immunochemical data demonstrated that these isozymes probably belong to the steroid-inducible gene family. Further characterisation of cyt. P-4507:3 indicated that it is a major inducible isozyme in man and that it metabolises the calcium entry blocker nifedipine but not a structurally similar drug nicardipine.

No description available.

Thesis (PhD)--Stellenbosch University, 2012. / Includes bibliography / ENGLISH ABSTRACT: Tuberculosis (TB) has been declared a global health emergency by the World Health Organisation, and consequently there is an urgency to develop improved methods of diagnosis and treatment. Despite the current TB epidemic, the disease can be treated effectively using isoniazid (INH) in combination with other antibiotics. However, INH is inactivated in the body by certain drug metabolising enzymes, which may reduce the efficacy of TB treatment. The activity of these drug metabolising enzymes, called NAT, are in turn reduced by nucleotide changes (SNPs) in the gene. These genetic variants (alleles) have been correlated with the rapid- (FA), intermediate- (IA), and slow acetylation (SA) enzymatic activity, and one is therefore able to investigate potential phenotypic effects via genotypic analyses. We investigated these genetic changes in the NAT1 and NAT2 genes in individuals from the local Coloured community (SAC) since this group has one of the highest TB incidences in the country. NAT2 is primarily responsible for the inactivation of INH, whilst NAT1 metabolises para-aminosalicyclic acid (PAS) which is used in the treatment of drug resistant TB. The NAT2 results indicated that the NAT2 alleles were not equally represented in three local ethnic groups studied, and subsequently the rapid, intermediate and slow acetylation activity reflected these differences. However, the relative frequency of these variants in the SAC and Caucasian groups were relatively low. These differences require further investigation to determine their overall relevance to the NAT2 activity differences between groups. In the case of the NAT1 analysis we also observed differences in the relative frequency of various NAT1 alleles between Caucasian and SAC individuals. However, many of these NAT1 SNPs and alleles have not as yet been characterised, so effects of these variants are currently unknown. Interestingly, the NAT1*4 and NAT1*10 alleles were the most prevalent NAT1 alleles in both Caucasians and SAC. The NAT1*4 allele exhibits the rapid NAT1 activity, whilst the activity of the NAT1*10 allele is currently subject to ongoing debate. In this respect, the analysis of NAT1 continues to be a topic for ongoing research. These results, observed for the NAT genes, underscore the importance of doing genetic analyses in local ethnic groups, since these differences may vary significantly between the groups. / AFRIKAANSE OPSOMMING: Tuberkulose (TB) is deur die Wêreldgesondheidsorganisasie (WGO) tot 'n globale gesondheidsnood verklaar en derhalwe is dit noodsaaklik dat nuwe, verbeterde diagnostiese metodes ontwikkel word, wat tot meer effektiewe behandeling kan lei. Ten spyte van die huidige TB-epidemie, kan die siekte doeltreffend behandel word deur middel van isoniasied (INH), in kombinasie te met ander antibiotika. INH kan egter geïnaktiveer word deur sekere ensieme in die liggaam, met die gevolg dat INH nie meer effektief is nie in die behandeling van TB. Die aktiwiteit van hierdie ensiem, die sogenaamde NAT2 (Arielamien N-asetieltransferase 2) ensiem, word op sy beurt beïnvloed deur sekere nukleotied veranderings (SNPs) in die geen. Hierdie genetiese veranderings gekorreleer met ensiemaktiwiteitsveranderings (geklassifiseer as vinnig (FA) Intermediêr (IA) en stadig (SA)), wat mens in staat stel om potensiële fenotipiese effekte te ondersoek deur middel van genotipiese analise. Ons het hierdie genetiese veranderings ondersoek in die NAT1 en NAT2 gene in individue van die Kleurling-gemeenskap (SAC) omdat díe bevolkingsgroep die hoogste voorkoms van TB in die land het. NAT2 is primêr verantwoordelik vir die inaktivering van INH, terwyl NAT1 para-amienosalisilaat (PAS) inaktiveer, wat gebruik word in die behandeling van midel-weerstandige TB. Die NAT2 resultate dui daarop dat die allele van die NAT2 geen nie eweredig verteenwoordig wasin die drie etniese groepe nie en derhalwe word die vinnige (FA), intermediêre (IA) en stadige (SA) ensiemaktiwiteite deur hierdie verskille weerspieël. Hoewel die teenwoordigheid van hierdie variante relatief laag was in die SAC en Koukasiër gemeenskappe, is verdere studies nodig om die omvang van hierdie verskille te bepaal ten onsigte van NAT2 aktiwiteit tussen groepe. In die geval van die NAT1 analise het ons verskille waargeneem in die voorkoms van verskeie NAT1 allele tussen Koukasiese en SAC individue. Baie van hierdie NAT1 SNPs is egter nog nie gekarakteriseer nie, en derhalwe is die effek van hierdie NAT1 variante onbekend. Die NAT1*4 en NAT1*10 allele was die prominentste NAT1 alleel in beide Koukasiërs en SAC. Die NAT1*4 is betrokke by vinnige NAT1 aktiwiteit, terwyl die effek van die NAT1*10 alleel nog onderhewig is aan aktiefwe debat. In hierdie verband, is die studie van NAT1 steeds 'n onderwerp vir toekomstige navorsing. Hierdie resultate, wat vir die NAT gene waargeneem is, beklemtoon die belangrikheid van verdere genetiese analises in plaaslike etniese groepe, aangesien hierdie verskille beduidend kan wees tussen die verskillende groepe.

The evolutionary history of the human species has generated complex patterns of population structure and linkage disequilibrium (non-random associations of alleles at different loci or LD). The understanding of these patterns is crucial to two of the most important challenges facing biomedical science today: the identification of disease predisposing genes and prediction of variable drug reactions. The genetic variation revealed by these endeavours can also illuminate the underlying population historical processes. Here, I illustrate each of these applications: first, by assessing the demographic context of cultural change in the British Isles. Y chromosome variation indicates that the Viking age invasions left a significant paternal legacy (at least in Orkney), while the Neolithic and Iron Age cultural transitions did not. In contrast, mitochondrial DNA and X chromosome variation indicate that one or more of these pre-Anglo-Saxon revolutions had a major effect on the maternal genetic heritage of the British Isles. Second, I provide conclusive evidence that diverse demographic histories produce strikingly different patterns of association. Elevated LD extends an order of magnitude further in the Lemba, a Bantu-Semitic hybrid population, than in the putative parental populations. A significant relationship between allele-frequency differentials in the parental populations and the Lemba LD demonstrates that it is admixture-generated. Third, I demonstrate that the genetic structure inferred in a heterogeneous sample using neutral markers (a) shows ethnic labels to be inaccurate descriptions of human population structure, and (b) predicts drug metabolising profiles, defined by the distribution of drug metabolising enzyme variants. Thus the trade-off between therapeutic response and adverse drug reactions will differ between different sub-clusters. Assessment of genetic structure during drug trials is therefore, like the empirical evaluation of each population’s pattern of LD, a necessity.