The EGR1 transactivator is overexpressed in prostate cancer, and its expression pattern suggests that EGR1 could potentially regulate a number of steps involved in initiation and progression of prostate cancer, such as mitogenesis, invasiveness, angiogenesis, and metastasis. To identify potential EGR1 target genes in an unbiased manner, we have utilized adenovirus-mediated expression of EGR1 in a prostate cancer cell line to identify specific genes that are induced by EGR1. Using oligonucleotide arrays, a number of EGR1-regulated genes were identified and their regulation was confirmed by quantitative reverse transcription-polymerase chain reaction analysis. One of the largest gene classes identified in this screen includes several neuroendocrine-associated genes (neuron-specific enolase, neurogranin), suggesting that EGR1 overexpression may contribute to the neuroendocrine differentiation that often accompanies prostate cancer progression. This screen also identified several growth factors such as insulin-like growth factor-II, platelet-derived growth factor-A, and transforming growth factor-␤1, which have previously been implicated in enhancing tumor progression. The insulin-like growth factor-II gene lies within the 11p15.5 chromosomal locus, which contains a number of other imprinted genes, and EGR1 expression was found to induce at least two other genes in this locus (IPL, p57 KIP2 ). Based on our results, coupling adenoviral overexpression with microarray and quantitative reverse transcription-polymerase chain reaction analyses could be a versatile strategy for identifying target genes of transactivators.

Aldose reductase enfolds NADP+/NADPH via a complex loop mechanism, with cofactor exchange being the rate-limiting step for the overall reaction. This study measures the binding constants of these cofactors in the wild-type enzyme, as well as a variety of active-site mutants (C298A, Y48H, Y48F, Y209F, H110A, W219A, and W20A), and seeks to identify the binding site and mechanism of the aldose reductase inhibitor alrestatin in the recombinant human enzyme. All the mutant enzymes, regardless of their enzyme activities, have normal or only slightly elevated coenzyme binding constants, suggesting a tertiary structure similar to that of the wild-type enzyme. Binding of alrestatin was detected by fluorescence assays, and by an ultrafiltration assay which measures the fraction of unbound alrestatin. Alrestatin binds preferentially to the enzyme/NADP+ complex, consistent with the steady-state inhibition pattern. Alrestatin binding and enzyme inhibition were abolished in the Tyr48 mutants Y48F and Y48H, implicating the positively charged anion well formed by the Asp43-/Lys77+/Tyr48(0)/NADP+ complex in inhibitor binding (Harrison et al., 1994; Bohren et al., 1994). The enzyme mutant W20A severely affected the inhibitory potencies of a variety of commercially developed aldose reductase inhibitors (zopolrestat, tolrestat, FK366, AL1576, alrestatin, ponalrestat, and sorbinil). Inhibition by citrate, previously shown to bind to the positively charged anion well, was not affected by this mutation. Inhibitors with flexible double aromatic ring systems (Zopolrestat, FK366, and ponalrestat) were less affected than others possessing a single aromatic ring system, suggesting that the additional pharmacophor ring system stabilizes the inhibitor by interaction at some other hydrophobic site.(ABSTRACT TRUNCATED AT 250 WORDS)

The essential identity of the fluorescence emission spectra of the remotely from the chromophore but in the folded protein may be intact proteins and the identity of the absorbance spectra of the situated in its vicinity. We conclude that the mutations influence isolated hexapeptides containing the chromophore further supthe fluorescence properties by changing the interactions between port the structural identity of the chromophores [14]. The evothe chromophore and its protein environment, lutionary relationship of the two GFPs has not been defined on a molecular level since only the amino acid sequence of Aequo-

The enzymes mainly responsible for ethanol degradation in humans are liver alcohol dehydrogenases (ADH) and aldehyde dehydrogenases (ALDH). Polymorphisms occur in both enzymes, with marked differences in the steady-state kinetic constants. The Km-values for ethanol of ADH isoenzymes relevant for alcohol degradation range from 49 microM to 36 microM, and the Vmax-values from 0.6 to 10 U/mg. Expression of an inactive form of the ALDH2 isoenzyme, the so-called Oriental variant, results in impaired acetaldehyde metabolizing capacity. The differences in ethanol and acetaldehyde metabolizing activities of allelic enzyme forms may be responsible in part for the large variation in the alcohol metabolism rate in humans. Interindividual differences in the isoenzyme pattern may contribute to the genetically determined predisposition for excessive alcohol intake.

The factors that govern blood and tissue concentrations of ethanol after its ingestion are the rate of absorption from the gastrointestinal tract, the space of distribution in the body, and the rate of elimination. Ethanol is absorbed rapidly by diffusion from the stomach and small intestines and is distributed in total body water. It is neither accumulated to any extent by specific organs nor preferentially bound to cellular components. It is eliminated almost entirely by oxidative metabolism in the liver. Consequently, after an initial equilibration phase, the primary determinant of the duration and extent of ethanol's pharmacologic and potentially pathologic effects is the rate of its oxidative metabolism. For this reason, the enzymatic pathways of ethanol metabolism and their control by genetic and environmental factors have been important areas for detailed study. This review focuses on recent gains in our knowledge of the biochemical genetics of alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH), the two principal enzymes of ethanol oxidation in liver, and discusses the relationship of the discovered genetic polymorphisms of these enzymes to individual differences in alcohol oxidizing capacity and to alcohol abuse liability. GENETIC INFLUENCE ON ETHANOL PHARMACOKINETICSIn the fasted state, ingested ethanol is absorbed principally from the duodenum and jejunum, owing to its rapid transit through an empty stomach.When ethanol is consumed with food or when the stomach is already full, substantial amounts of ethanol are absorbed from the stomach. As much as 70% of the ingested ethanol can be From the absorbed from the stomach under these conditions and absorption may not be completed until 4 to 5 hours after ingestion.' Foodstuffs such as fats and hypertonic solutions delay gastric emptying. Delayed absorption leads to a lesser amount of ethanol appearing in the systemic circulation, because there is more time for first-pass metabolism to occur.2 This is the metabolism of ethanol by enzymes in the gastric and intestinal mucosa and the liver, as ethanol is absorbed and passes via the portal circulation through the liver to the systemic circulation. The rate of ethanol absorption is partially determined by genetic factor^,^ although the mechanism for this effect is unknown.After the absorptive and distributive phases are complete, rates of ethanol elimination can be estimated by measuring ethanol concentrations in the blood or breath over time. There is still lack of general agreement on what are the best ways to perform such calculations, because ethanol can be metabolized by different enzyme systems that have different affinities for ethanol (vide infra). However, experimental studies in humans can be performed only over a limited range of ethanol concentrations with safety. Under these circumstances, the rate of disappearance from blood or breath in the postabsorption-distribution phase can usually be described adequately by a single compartment model with pseudozero order (Widmark) or single K, Mic...