Activation of LINE-1 Retrotransposon Increases the Risk of Epithelial-Mesenchymal Transition and Metastasis in Epithelial Cancer.

Epithelial cancers comprise 80-90% of human cancers. During the process of cancer progression, cells lose their epithelial characteristics and acquire stem-like mesenchymal features that are resistant to chemotherapy. This process, termed the epithelial-mesenchymal transition (EMT), plays a critical role in the development of metastases. Because of the unique migratory and invasive properties of cells undergoing the EMT, therapeutic control of the EMT offers great hope and new opportunities for treating cancer. In recent years, a plethora of genes and noncoding RNAs, including miRNAs, have been linked to the EMT and the acquisition of stem cell-like properties. Despite these advances, questions remain unanswered about the molecular processes underlying such a cellular transition. In this article, we discuss how expression of the normally repressed LINE-1 (or L1) retrotransposons activates the process of EMT and the development of metastases. L1 is rarely expressed in differentiated stem cells or adult somatic tissues. However, its expression is widespread in almost all epithelial cancers and in stem cells in their undifferentiated state, suggesting a link between L1 activity and the proliferative and metastatic behaviour of cancer cells. We present an overview of L1 activity in cancer cells including how genes involved in proliferation, invasive and metastasis are modulated by L1 expression. The role of L1 in the differential expression of the let-7 family of miRNAs (that regulate genes involved in the EMT and metastasis) is also discussed. We also summarize recent novel insights into the role of the L1-encoded reverse transcriptase enzyme in epithelial cell plasticity that suggest it might be a potential therapeutic target that could reverse the EMT and the metastasis-associated stem cell-like properties of cancer cells.

The impact of glutathione transferase kappa deficiency on adiponectin multimerisation in vivo.

Glutathione transferase Kappa (GSTK1-1) also termed disulfide bond-forming oxidoreductase A-like protein (DsbA-L) has been implicated in the post-translational multimerization of adiponectin and has been negatively correlated with obesity in mice and humans. We investigated adiponectin in Gstk1(-/-) mice and surprisingly found no difference in the levels of total serum adiponectin or the level of high molecular weight (HMW) multimers when compared with normal controls. Non-reducing SDS-polyacrylamide gel electrophoresis and western blotting also showed a similar distribution of low, middle and HMW multimers in normal and Gstk1(-/-) mice. Variation in adiponectin has been correlated with glucose tolerance and with the levels of phosphorylated AMP-kinase but we found similar glucose tolerance and similar levels of phospho 5-AMP-activated protein kinase in normal and Gstk1(-/-) mice. Consequently, our findings suggest that GSTK1-1 is not absolutely required for adiponectin multimerization in vivo and alternate pathways may be activated in GSTK1-1 deficiency.

One-solvent polymorph screen of carbamazepine.

To emphasize the fact that solvents can be either critical or immaterial in crystallizing specific polymorphs, a method for obtaining multiple polymorphs of a compound using only one solvent is demonstrated. By varying the crystallization temperature and level of supersaturation, three of the four polymorphs of carbamazepine (CBZ; 5H-dibenz [b,f]azepine-5-carboxamide) were crystallized from cumene (isopropyl benzene). Form III, also referred to as the primitive monoclinic form, was produced at temperatures below 60 degrees C from supersaturated solutions concentrated at less than twice the solubility of that form. When the supersaturation was increased to twice the solubility of form III at temperatures below 60 degrees C, form II, also referred to as the trigonal form, was produced. Form I, also referred to as the triclinic form, was produced regardless of the level of supersaturation at temperatures above 80 degrees C. Between 60 degrees C and 80 degrees C, mixtures of forms were produced. Competition slurries were employed to establish the transition temperature to be between 79 degrees C and 82 degrees C for the enantiotropically related forms III and I. These results indicate that crystallization of CBZ from cumene can either be under thermodynamic control or affected by the kinetics of crystallization of metastable forms. This raises the question about the importance of solvent diversity when looking for polymorphs, suggesting that a rational experimental design can be used to greatly reduce the number of solvents and crystallization conditions. The results of this one-solvent polymorph screen correlate somewhat with a phase-solubility diagram for CBZ.

GSTZ1d: a new allele of glutathione transferase zeta and maleylacetoacetate isomerase.

The zeta class glutathione transferases (GSTs) are known to catalyse the isomerization of maleylacetoacetate (MAA) to fumarylacetoacetate (FAA), and the biotransformation of dichloroacetic acid to glyoxylate. A new allele of human GSTZ1, characterized by a Thr82Met substitution and termed GSTZ1d, has been identified by analysis of the expressed sequence tag (EST) database. In European Australians, GSTZ1d occurs with a frequency of 0.16. Like GSTZ1b-1b and GSTZ1c-1c, the new isoform has low activity with dichloroacetic acid compared with GSTZ1a-1a. The low activity appears to be due to a high sensitivity to substrate inhibition. The maleylacetoacetate isomerase (MAAI) activity of all known variants was compared using maleylacetone as a substrate. Significant differences in activity were noted, with GSTZ1a-1a having a notably lower catalytic efficiency. The unusual catalytic properties of GSTZ1a-1a in both reactions suggest that its characteristic arginine at position 42 plays a significant role in the regulation of substrate access and/or product release. The different amino acid substitutions have been mapped on to the recently determined crystal structure of GSTZ1-1 to evaluate and explain their influence on function.

Crystal structure of maleylacetoacetate isomerase/glutathione transferase zeta reveals the molecular basis for its remarkable catalytic promiscuity.

Maleylacetoacetate isomerase (MAAI), a key enzyme in the metabolic degradation of phenylalanine and tyrosine, catalyzes the glutathione-dependent isomerization of maleylacetoacetate to fumarylacetoacetate. Deficiencies in enzymes along the degradation pathway lead to serious diseases including phenylketonuria, alkaptonuria, and the fatal disease, hereditary tyrosinemia type I. The structure of MAAI might prove useful in the design of inhibitors that could be used in the clinical management of the latter disease. Here we report the crystal structure of human MAAI at 1.9 A resolution in complex with glutathione and a sulfate ion which mimics substrate binding. The enzyme has previously been shown to belong to the zeta class of the glutathione S-transferase (GST) superfamily based on limited sequence similarity. The structure of MAAI shows that it does adopt the GST canonical fold but with a number of functionally important differences. The structure provides insights into the molecular bases of the remarkable array of different reactions the enzyme is capable of performing including isomerization, oxygenation, dehalogenation, peroxidation, and transferase activity.

Identification of novel glutathione transferases and polymorphic variants by expressed sequence tag database analysis.

The human expressed sequence tag (EST) database can be searched by different sequence alignment strategies to identify new members of gene families and allelic variants. To illustrate the value of database analysis for gene discovery, we have focused on the glutathione S-transferase (GST) super family, an approach that has led to the identification of the Zeta class. The Zeta class GSTs catalyze the glutathione-dependent biotransformation of alpha-haloacids and the isomerization of maleylacetoacetic acid to fumarylacetoacetic acid, an essential step in the catabolism of tyrosine. Allelic variants of the GST Z1 and GST A2 genes have also been identified by EST database analysis. One GST Z1 variant (GST Z1A) has significantly higher activity with dichloroacetic acid as a substrate than other GST Z1 isoforms. This variant may be important in the clinical treatment of lactic acidosis where dichloroacetic acid is prescribed. Our experience with the application of EST database searching methods suggests that it may be productively applied to other gene families of pharmacogenetic interest.

Polymorphism- and species-dependent inactivation of glutathione transferase zeta by dichloroacetate.

Glutathione transferase zeta catalyzes the glutathione-dependent oxidation or conjugation of a range of alpha-haloacids. Repeated administration of dichloroacetate to human subjects increases its plasma elimination half-life, and the activity of glutathione transferase zeta is decreased in rats given dichloroacetate. The objective of the studies presented here was to investigate the kinetics and mechanism of the dichloroacetate-induced decrease in glutathione transferase zeta activity. The rate constants (k(inact)) for the dichloroacetate-dependent inactivation of glutathione transferase zeta in liver cytosol are in the following order: rat > mouse > human; the half-maximal inhibitory concentration (K(inact)) of DCA did not differ among the species that were studied. In contrast to dichloroacetate, chlorofluoroacetate produced much less inactivation of mouse liver glutathione transferase zeta activity. Moreover, the addition of N-acetyl-L-cysteine or potassium cyanide did not fully block the dichloroacetate-induced inactivation of glutathione transferase zeta. The k(inact) values for the dichloroacetate-induced inactivation of four polymorphic variants of recombinant human glutathione transferase zeta (hGSTZ1-1) were in the following order: variant 1a-1a < 1b-1b approximately 1c-1c approximately 1d-1d. The dichloroacetate-induced inactivation of hGSTZ1-1 was irreversible. The binding of radioactivity from [1-(14)C]dichloroacetate and from [(35)S]glutathione to recombinant hGSTZ1c-1c was demonstrated, indicating covalent modification of the protein. These results show that dichloroacetate is a mechanism-based inactivator of glutathione transferase zeta and is biotransformed to electrophilic metabolites that covalently modify and, thereby, inactivate the enzyme.

Discovery of a functional polymorphism in human glutathione transferase zeta by expressed sequence tag database analysis.

Analysis of the expressed sequence tag (EST) database by sequence alignment allows a rapid screen for polymorphisms in proteins of physiological interest. The human zeta class glutathione transferase GSTZ1 has recently been characterized and analysis of expressed sequence tag clones suggested that this gene may be polymorphic. This report identifies three GSTZ1 alleles resulting from A to G transitions at nucleotides 94 and 124 of the coding region, GSTZ1*A-A94A124; GSTZ1*B-A94G124; GSTZ1*C-G94G124. Polymerase chain reaction/restriction fragment length polymorphism analysis of a control Caucasian population (n = 141) showed that all three alleles were present, with frequencies of 0.09, 0.28 and 0.63 for Z1*A, Z1*B and Z1*C, respectively. These nucleotide substitutions are non-synonymous, with A to G at positions 94 and 124 encoding Lys32 to Glu and Arg42 to Gly substitutions, respectively. The variant proteins were expressed in Escherichia coli as 6X His-tagged proteins and purified by Ni-agarose column chromatography. Examination of the activities of recombinant proteins revealed that GSTZ1a-1a displayed differences in activity towards several substrates compared with GSTZ1b-1b and GSTZ1c-1c, including 3.6-fold higher activity towards dichloroacetate. This report demonstrates the discovery of a functional polymorphism by analysis of the EST database.

Protein carbonyl formation on mucosal proteins in vitro and in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease, but few studies have investigated protein oxidation in the inflamed mucosa. In this study, protein carbonyl formation on colonic mucosal proteins from mice was investigated following in vitro exposure of homogenates to iron/ascorbate, hydrogen peroxide, hypochloric acid (HOCl), or nitric oxide (*NO). Total carbonyl content was measured spectrophotometrically by derivatization with dinitrophenylhydrazine (DNPH), and oxidation of component proteins within the tissue was examined by Western blotting for DNPH-derivatized proteins using anti-dinitrophenyl DNP antibodies. These results were compared with protein carbonyl formation found in the acutely inflamed mucosa from mice with colitis induced by dextran sulfate sodium (DSS) administered at 5% w/v in the drinking water for 7 d. In vitro, carbonyl formation was observed after exposure to iron/ascorbate, HOCl and *NO. Iron/ascorbate (20 microM/20 mM) exposure for 5 h increased carbonyl groups by 80%, particularly on proteins of 48, 75-100, 116, 131, and 142 kDa. Oxidation by 0.1 and 0.5 mM HOCl did not increase total carbonyl levels, but Western blotting revealed carbonyl formation on many proteins, particularly in the 49-95 kDa region. After exposure to 1-10 mM HOCl, total carbonyl levels were increased by 0.5 to 12 times control levels with extensive cross-linking and fragmentation of proteins rich in carbonyl groups observed by Western blotting. In mice with acute colitis induced by DSS, protein carbonyl content of the inflamed mucosa was not significantly different from control mucosa, (7.80 +/- 1.05 vs. 8.43 +/- 0.59 nmo/mg protein respectively, p = .16 n = 8, 10); however, Western blotting analysis indicated several proteins of molecular weight 48, 79, 95, and 131 kDa that exhibited increased carbonyl content in the inflamed mucosa. These proteins corresponded to those observed after in vitro oxidation of normal intestinal mucosa with iron/ ascorbate and HOCl, suggesting that both HOCl and metal ions may be involved in protein oxidation in DSS-induced colitis. Identification and further analysis of the mucosal proteins susceptible to carbonyl modification may lead to a better understanding of the contribution of oxidants to the colonic mucosa tissue injury in inflammatory bowel disease.

Design and synthesis of novel quinoxaline-2,3-dione AMPA/GlyN receptor antagonists: amino acid derivatives.

PNQX (1,4,7,8,9,10-hexahydro-9-methyl-6-nitropyrido[3, 4-f]quinoxaline-2,3-dione) is a potent AMPA (IC50 = 0.063 microM) and GlyN (IC50 = 0.37 microM) receptor antagonist that was developed in our laboratories. While possessing a desirable in vitro and in vivo activity profile, this compound suffers from low aqueous solubility. In an effort to improve its potency and physical properties, we have designed and synthesized novel ring-opened analogues 4, 6, 9, and 11. Modeling analyses demonstrated that, while the 5-substituent in these analogues was forced to adopt an out-of-plane conformation due to steric contacts with neighboring substituents, the overall structure retained a good fit to a previously described AMPA pharmacophore model. This nonplanar orientation may lessen efficient packing in the solid state, compared to PNQX, leading to increased water solubility. Indeed, several nonplanar analogues containing appropriate functionalities, for example, the sarcosine analogue 9, were found to retain AMPA (IC50 = 0.14 microM) and GlyN (IC50 = 0.47 microM) receptor affinity and possess improved aqueous solubility compared to PNQX. The synthesis and the SAR of these compounds are discussed.

Parkinson's disease, pesticides, and glutathione transferase polymorphisms.

BACKGROUND: Parkinson's disease is thought to be secondary to the presence of neurotoxins, and pesticides have been implicated as possible causative agents. Glutathione transferases (GST) metabolise xenobiotics, including pesticides. Therefore, we investigated the role of GST polymorphisms in the pathogenesis of idiopathic Parkinson's disease. METHODS: We genotyped by PCR polymorphisms in four GST classes (GSTM1, GSTT1, GSTP1, and GSTZ1) in 95 Parkinson's disease patients and 95 controls. We asked all patients for information about pesticide exposure. FINDINGS: The distribution of the GSTP1 genotypes differed significantly between patients and controls who had been exposed to pesticides (controls vs patients: AA 14 [54%] of 26 vs seven [18%] of 39; AB 11 [42%] of 26 vs 22 [56%] of 39; BB 1 [4%] of 26 vs six [15%] of 39; AC 0 vs four [10%] of 39, p=0.009). No association was found with any of the other GST polymorphisms. Pesticide exposure and a positive family history were risk factors for Parkinson's disease. INTERPRETATION: GSTP1-1, which is expressed in the blood-brain barrier, may influence response to neurotoxins and explain the susceptibility of some people to the parkinsonism-inducing effects of pesticides.

Hydrogen-bonding patterns in a centro-symmetric structure with Z' = 2: alpha, alpha', alpha''-trimethyl-1,3,5-benzenetrimethanol.

The title molecule, C12H18O3, crystallized in the centrosymmetric space group P2(1)/c with two molecules in the asymmetric unit. Each molecule donates three, and accepts three, hydrogen bonds. The Od...Oa distances in these bonds range from 2.687(3) to 2.787(2) A. The hydroxyl H atoms are ordered. A three-dimensional network of hydrogen-bond chains is formed which is 'decorated' with sets of cyclic hydrogen bonds and numerous finite hydrogen-bond patterns. This structure shows both similarities and differences with respect to the structures of the related molecules benzene-1,3,5-triacetic acid and benzene-1,3,5-trimethanol.

Salicylate hydroxylation as an indicator of hydroxyl radical generation in dextran sulfate-induced colitis.

Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease. This study investigated hydroxyl radical (.OH) generation in the inflamed colon of dextran sulfate sodium (DSS)-induced colitis by measuring the .OH-specific product of salicylate hydroxylation, 2,3-dihydroxybenzoic acid (DHB). Colitis was induced in 6-7 week old CBA/H male mice by supplementing the drinking water with 5% DSS for 7 days. On the last day of dextran exposure, mice were injected with salicylate (SAL) (100 mg/kg i.p.) 60 min before sacrifice, and mucosal homogenates were assayed for SAL and 2,3-DHB by HPLC with fluorescence and electrochemical detection. Mucosal 2,3-DHB levels in mice exposed to 5% DSS were increased by 83% (p < .005); however, SAL levels were also elevated by 182% (p < .001). This translated to a 34% decrease in the ratio 2,3-DHB:SAL in inflamed mucosa, possibly indicating greater catabolism or decreased production of 2,3-DHB. In vitro investigation of the stability of DHBs and SAL in the presence of oxidants of inflammatory lesions revealed that 2,3-DHB and 2,5-DHB were rapidly degraded by hypochlorous acid (HOCl), with initial decomposition rates of 190 and 281 nmol/min, respectively (100microM DHB with 200microM HOCl). Methionine prevented decomposition of DHBs in vitro; however, in mice with 5% DSS-induced colitis, where mucosal myeloperoxidase activity was ten-fold control levels (p < .001), administration of methionine (up to 200 mg/kg i.p.) with SAL was ineffective at increasing the ratio 2,3-DHB:SAL. SAL was also degraded in vitro by HOCl (4.7 nmol/min) resulting in the formation of new fluorescent species which may be useful as indicators of HOCl-mediated injury. Salicylate hydroxylation was unable to provide conclusive evidence supporting a role for .OH in the tissue injury of DSS-induced colitis, as metabolic disturbances in the diseased animals other than changes in .OH generation may have altered 2,3-DHB levels. This problem is relevant to any study involving the in vivo use of trapping molecules. In particular, the susceptibility of 2,3-DHB to degradation by HOCl brings into question the usefulness of salicylate hydroxylation for measurement of .OH-generation in any neutrophilic inflammatory lesion.

Characterization and chromosome location of the gene GSTZ1 encoding the human Zeta class glutathione transferase and maleylacetoacetate isomerase.

The Zeta class of cytosolic glutathione-S-transferases (GSTs) has recently been identified and spans a range of species from plants to humans. The cDNA and protein of a human member of this class have been previously characterised in our laboratory. This cDNA has also been described as maleylacetoacetate isomerase (MAAI), an enzyme of the phenylalanine catabolism pathway (Fernandez-Canon and Penalva, 1998). The present study has determined the structure and chromosome location of the gene encoding human GSTZ1/MAAI. The gene spans approximately 10.9 kb and is composed of 9 exons. Three intron positions of GSTZ1 were precisely conserved compared to the carnation and Caenorhabditis elegans Zeta GST genes. Fluorescent in situ hybridization mapped the gene to a single locus on chromosome 14q24.3, which is in agreement with an independent localization between the Genethon markers D14S263 and D14S67. The coding region of the gene differed from the GSTZ1 cDNA at two nucleotide positions in exon 3, resulting in Lys-32-->Glu and Arg-42--> Gly substitutions. This gene structure information will allow analysis of the polymorphism in genomic DNA samples, and enables further investigations into genetic defects in this step of the phenylalanine catabolism pathway.

1,4-dimethoxy-2-naphthoic acid.

The title compound, C13H12O4, crystallized in the centrosymmetric space group P2(1)/c and exhibits cyclic dimer hydrogen bonding about a center of symmetry. The carboxylic H atom is modeled as disordered over two half-occupancy sites. Overall, the hydrogen bonding is little affected by the methoxyl groups and is very similar to that in 2-naphthoic acid.

5,8-Dimethoxy-1-naphthoic acid and methyl 5,8-dimethoxy-1-naphthoate.

In 5,8-dimethoxy-1-naphthoic acid, C13H12O4, hydrogen bonding is of the cyclic dimer type. The acid H atom is modelled as being distributed equally over two sites. In addition to the conventional hydrogen bonds, there are three significantly attractive C-H...O interactions. The dihedral angle between the naphthalene core plane and the carboxyl plane is 80.0 (1) degrees. In methyl 5,8-dimethoxy-1-naphthoate, C14H14O4, there are no conventional hydrogen bonds but there are three significantly attractive C-H...O interactions. With the exception of the C-O distances in the carboxyl groups, the molecular geometries of the acid and the ester are quite similar.

Colonic antioxidant status in dextran sulfate-induced colitis in mice.

SUMMARY: : Reactive oxygen and nitrogen species have been implicated as mediators of mucosal injury in inflammatory bowel disease (IBD). This study investigated the status of the endogenous antioxidants and markers of oxidative mucosal injury in dextran sulfate-induced colitis in mice. Colitis was induced by supplementing the drinking water with 5% dextran sulfate. After 8 days of dextran treatment, the colonic mucosa was analyzed for total radical scavenging capacity, major lipophilic and aqueous antioxidants, and thiol-containing markers of oxidative injury. Compared with control mucosa, there was a 3.3-fold increase in mucosal myeloperoxidase activity (p < 0.001), corresponding to the neutrophil infiltration seen histologically. Significant decreases in total peroxyl radical scavenging capacity (15.7%, p < 0.05) and mucosal antioxidant levels, including ubiquinol-9 and ascorbate, were found (53.1 and 17.6%, respectively, p < 0.001). In contrast, α-tocopherol and urate levels were increased by 63.7 and 109%, respectively (p < 0.001). Glyceraldehyde-3-phosphate dehydrogenase activity, previously shown to be inactivated by thiol oxidation in inflamed but not in noninflamed IBD epithelium, and total reduced thiol content were also significantly decreased by 33.8 and 26.3%, respectively (p < 0.001). These results parallel those reported in IBD mucosa, strengthening the relevance of dextran sulfate-induced colitis in mice to IBD and supporting the use of this model to provide insights into the pathogenesis of oxidative mucosal injury and the development of novel therapeutic strategies.

1,4,5,8-naphthalenetetracarboxylic acid cyclic 1,8-anhydride bis(dimethyl sulfoxide) solvate and 1,4,5,8-naphthalenetetracarboxylic 1,8:4,5-dianhydride.

1,4,5,8-Naphthalenetetracarboxylic acid cyclic 1,8-anhydride crystallized from dimethyl sulfoxide (DMSO) as the solvate, C14H6O7.2C2H6OS, in the centrosymmetric space group P1. Two O-H...O hydrogen bonds with O...O distances of 2.592 (3) and 2.598 (3) A are formed, with the two carboxylic acid OH groups as donors and the O atoms of the two inequivalent DMSO molecules as acceptors. The carboxylic H atoms are ordered, as are the carboxylic O atoms. In addition to the conventional hydrogen bonds, there are numerous C-H...O interactions consistent with the large number of potential CH donors and O-atom acceptors. 1,4,5,8-Naphthalenetetracarboxylic 1,8:4,5-dianhydride, C14H4O6, crystallized in the centrosymmetric space group P2(1)/c with half the molecule as the asymmetric unit. Each molecule is involved in four significant C-H...O interactions as a donor and in an additional four as an acceptor. These eight interactions link each molecule to six neighboring molecules, forming a three-dimensional network. Geometric parameters of both substances are in general agreement with analogous parameters for naphthalic anhydride, monosodium 1,4,5,8-naphthalenetetracarboxylic acid cyclic 1,8-anhydride monohydrate and 1,4,5,8-naphthalenetetracarboxylic 1,8:4,5-dianhydride, as reported previously.

2-Naphthoic acid at 153 K.

The structure of 2-naphthoic acid, C11H8O2, has been investigated at 153 K in order to determine the degree of disorder of the carboxylic acid group for comparison with that of the room-temperature structure. Analysis of the anisotropic displacement ellipsoids of the carboxyl-O atoms demonstrated that these parameters are wholly consistent with thermal motion of the O atoms. A model with ordered carboxyl-O atoms, but with the acid-H atom refined at two sites with 0.5 occupancy at each, was found to be statistically significantly better than a model with ordered carboxyl-O atoms and an ordered acid-H atom. Thus, as in the room-temperature study, the best structural description is that the O atoms are ordered and the acid-H atom is disordered. Nonetheless, comparisons of the geometric parameters of the carboxyl groups at 296 and 153 K suggest progress toward a fully ordered structure at the lower temperature. Except for the expected slight overall contraction and the slightly altered geometry of the carboxyl group, the structure is virtually the same as at room temperature.