1. An important feature of the work of many molecular biologists is identifying which genes are switched on and off in a cell under different environmental conditions or subsequent to xenobiotic challenge. Such information has many uses, including the deciphering of molecular pathways and facilitating the development of new experimental and diagnostic procedures. However, the student of gene hunting should be forgiven for perhaps becoming confused by the mountain of information available as there appears to be almost as many methods of discovering differentially expressed genes as there are research groups using the technique. 2. The aim of this review was to clarify the main methods of differential gene expression analysis and the mechanistic principles underlying them. Also included is a discussion on some of the practical aspects of using this technique. Emphasis is placed on the so-called 'open' systems, which require no prior knowledge of the genes contained within the study model. Whilst these will eventually be replaced by 'closed' systems in the study of human, mouse and other commonly studied laboratory animals, they will remain a powerful tool for those examining less fashionable models. 3. The use of suppression-PCR subtractive hybridization is exemplified in the identification of up- and down-regulated genes in rat liver following exposure to phenobarbital, a well-known inducer of the drug metabolizing enzymes. 4. Differential gene display provides a coherent platform for building libraries and microchip arrays of 'gene fingerprints' characteristic of known enzyme inducers and xenobiotic toxicants, which may be interrogated subsequently for the identification and characterization of xenobiotics of unknown biological properties.
(1987). The Effect of Peroxisome Prouferators on Microsomal. Peroxisomal, and Mitochondrial Enzyme Activities in the Liver and Kidney. Drug Metabolism Reviews: Vol. 18, No. 4, pp. 441-515.
Abstract Six species (CD‐1 mouse, Fischer 344 rat, Syrian golden hamster, Duncan‐Hartley guinea pig, half‐lop rabbit and marmoset monkey) were treated orally with ciprofibrate, a potent oxyisobutyrate hypolipidaemic drug for 14 days. A dosedependent liver enlargement was observed in the mouse and rat and at the high dose level in the hamster. A marked dose‐dependent increase in the 12‐hydroxylation of lauric acid was observed in the treated mouse, hamster, rat, and rabbit, associated with a concomitant elevation in the specific content of cytochrome P‐450 4A1 apoprotein, determined by an ELISA technique. Similarly, in these responsive species, an increase in mRNA levels coding for cytochrome P450 4A1 was observed. Lauric acid 12‐hydroxylation was unchanged in the guinea pig and marmoset after ciprofibrate pre‐treatment, and cytochrome P‐450 4A1 was not detected immunochemically in liver microsomes from these latter species. In the untreated mouse, hamster, rat, and rabbit, the 12‐hydroxylation of lauric acid was more extensive than the 11‐hydroxylation, whereas in the guinea pig and marmoset the activity ratios were reversed, with 11‐hydroxylation predominating. Peroxisomal fatty acid β‐oxidation was markedly induced in the mouse, hamster, rat, and rabbit on treatment at the higher dose level (39‐, 3‐, 13‐ and 5‐fold, respectively) and was slightly increased in the marmoset (2‐fold), yet was unchanged in the guinea pig following treatment. In the marmoset the increase in peroxisomal β‐oxidation was 3‐ to 4‐fold at the high dose level; however, the dose levels used in the marmoset were 20 and 100 mg/kg as opposed to 2 and 20 mg/kg in the other species. The differences in the foregoing hepatic enzyme responses to ciprofibrate between the species examined in our studies indicate a specific pattern of enzyme changes in responsive species. In the responsive species (rat, mouse, hamster, and rabbit), cytochrome P‐450 4A1 specific content and related enzyme activity were increased concomitant with elevated peroxisomal β‐oxidation. By contrast, the marmoset and guinea pig lack the coordinate hepatic induction of peroxisomal and microsomal parameters and may be categorized as less responsive species. Accordingly, the rat hepatic responses to peroxisome proliferators cannot confidently be used to predict biological responses in primates, with obvious implications for the extrapolation of animal data to man.
Objectives We have identified a member of the karyopherin (importin) α family of nuclear import factors as being modulated in rat liver following exposure to the hypolipidaemic and liver growth agent Wy-14,643. To examine the hypothetical role of this protein family as a checkpoint in receptor-mediated signalling, we characterized the rat karyopherin α (Kpna) gene family and present cDNA sequences and gene structures for all six rat Kpna genes. Further, we have assembled a comprehensive panel of Kpna coding regions from a range of metazoa, which we have subjected to phylogenetic analysis: This represents by far the most complete phylogenetic study of metazoan karyopherins, including several evolutionary intermediates not previously examined. The phylogeny reveals three Kpna subfamilies with distinct, conserved gene structures, shedding light on the evolutionary origins of this multigene family in metazoa. Methods and results Using quantitative PCR, we have analysed Kpna transcript levels in 44 rat tissues; Kpna transcripts show a wide variation in their distribution both in absolute and relative terms, suggestive of specialized roles for each member. We also demonstrate that Kpna genes are regulated in rat liver and isolated hepatocytes in a xenobiotic-specific manner for a number of chemically distinct liver growth agents. Conclusions In light of the crucial role of nuclear import in mediating the genomic changes elicited through nuclear receptor activation, we postulate that changes in the levels of specific karyopherins α during xenobiotic-mediated liver growth represent an important component of the cellular response to the external stimuli that trigger these events.
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