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R Lindahl

Publications and source records attributed to R Lindahl.

At least 19 recordsLinked to original sources
PubMed2001

Increase in class 2 aldehyde dehydrogenase expression by arachidonic acid in rat hepatoma cells.

Aldehyde dehydrogenase (ALDH) is a family of several isoenzymes important in cell defence against both exogenous and endogenous aldehydes. Compared with normal hepatocytes, in rat hepatoma cells the following changes in the expression of ALDH occur: cytosolic class 3 ALDH expression appears and mitochondrial class 2 ALDH decreases. In parallel with these changes, a decrease in the polyunsaturated fatty acid content in membrane phospholipids occurs. In the present study we demonstrated that restoring the levels of arachidonic acid in 7777 and JM2 rat hepatoma cell lines to those seen in hepatocytes decreases hepatoma cell growth, and increases class 2 ALDH activity. This latter effect appears to be due to an increased gene transcription of class 2 ALDH. To account for this increase, we examined whether peroxisome-proliferator-activated receptors (PPARs) or lipid peroxidation were involved. We demonstrated a stimulation of PPAR expression, which is different in the two hepatoma cell lines: in the 7777 cell line, there was an increase in PPAR alpha expression, whereas PPAR gamma expression increased in JM2 cells. We also found increased lipid peroxidation, but this increase became evident at a later stage when class 2 ALDH expression had already increased. In conclusion, arachidonic acid added to the culture medium of hepatoma cell lines is able to partially restore the normal phenotype of class 2 ALDH, in addition to a decrease in cell growth.

Aldehyde Dehydrogenase↗
PubMed2001

Beyond the catalytic core of ALDH: a web of important residues begins to emerge.

Site-directed mutagenesis was performed in class 3 aldehyde dehydrogenase (ALDH) on both strictly conserved, non-glycine residues, Glu-333 and Phe-335. Both lie in Motif 8 and are indicated to be of central catalytic importance from their positions in the tertiary structure. In addition, a highly conserved residue at the end of Motif 8, Pro-337, and Asp-247, which interacts with the main chain of Motif 8, were also mutated. All substitutions were conservative. Kinetic values clearly show that Glu-333 and Phe-335 are crucial to efficient catalysis, along with Asp-247. Pro-337 appears to have a different role, most likely relating to folding.

Aldehyde Dehydrogenase↗
PubMed2001

Coenzyme specificity in aldehyde dehydrogenase.

Influences on coenzyme preference are explored. Lysine 137 (192 in class 1/2 ALDH) lies close to the adenine ribose, directly interacting with the adenine ribose in NAD-specific ALDHs and the 2'-phosphate of NADP in NADP-specific ALDHs. Lys-137 in class 3 ALDH interacts with the adenine ribose indirectly through an intervening water molecule. However, this residue is present in all ALDHs and, as a result, is unlikely to directly influence coenzyme specificity. Glutamate 140 (195) coordinates the 2'- and 3'-hydroxyls of the adenine ribose of NAD in the class 3 tertiary structure. Thus, it appeared that this residue would influence coenzyme specificity. Mutation to aspartate, asparagine, glutamine or threonine shifts the coenzyme specificity towards NADP, but did not completely change the specificity. Still, the mutants show the 2'-phosphate of NADP is repelled by Glu-140 (195). Although Glu-140 (195) has a major influence on coenzyme specificity, it is not the only influence since class 3 ALDHs, can use both coenzymes, and class 2 ALDHs, which are NAD-specific, have a glutamate at this position. One explanation may be that the larger space between Lys-137 (192) and the adenine ribose hydroxyls in the class 3 ALDH:NAD binary structure may provide space to accommodate the 2'-phosphate of NADP. Also, a structural shift upon binding NADP may also occur in class 3 ALDHs to help accommodate the 2'-phosphate of NADP.

Aldehyde Dehydrogenase↗
PubMed2001

Aldehyde dehydrogenase 3 gene regulation: studies on constitutive and hypoxia-modulated expression.

We have previously shown that expression of the Class 3 aldehyde dehydrogenase gene (ALDH3) is abrogated by hypoxia. This phenomenon occurs in rat hepatoma systems in which ALDH3 expression is xenobiotic-inducible as well as in rat primary corneal epithelial cells that exhibit high constitutive ALDH3 expression. We have begun to test various segments of the ALDH3 5' flanking region for elements that may mediate this effect using CAT reporter gene constructs. In addition, although the involvement of the Ah receptor nuclear translocator (ARNT) in xenobiotic induction of ALDH3 is well established, the role of ARNT in constitutive ALDH3 expression is not clear. Moreover, ARNT is also a component of the hypoxia inducible factor-1 (HIF-1) bipartite transcription factor complex that mediates hypoxic induction of a variety of genes. Concomitant activation of the xenobiotic and hypoxia pathways results in cross-talk and functional interference. It has been hypothesized that this interference is due to limiting levels of ARNT. To examine if ARNT levels are limiting during hypoxic and xenobiotic induction in the context of ALDH3 expression and to examine possible roles of ARNT in constitutive expression of ALDH3 in corneal epithelial cells we co-transfected rat corneal epithelial cells and H4-II-EC3 rat hepatoma cells with ALDH3 5' UTR-CAT reporter genes and expression vectors containing either wild type or dominant negative forms of ARNT. Our results indicate that during hypoxia and xenobiotic induction of ALDH3 in H4-II-EC3 cells ARNT is not the limiting transcription factor. Further, neither wild type nor dominant negative ARNT had effects on constitutive ALDH3 expression in corneal epithelial cells.

Aldehyde Dehydrogenase↗
PubMed2001

Determination of morpholine in air by derivatisation with 1-naphthylisothiocyanate and HPLC analysis.

A method for the determination of morpholine in air was developed. Samples were collected with adsorbent tubes containing XAD-2 resin coated with 1-naphthylisothiocyanate (NIT). The thiourea derivative formed was subsequently desorbed with acetonitrile and analysed by HPLC with UV detection. The recovery after gas phase spiking with morpholine (2.2-1570 micrograms) was 91% (86-100%) with a relative standard deviation of 5.5%. No effect on recovery from relative humidity or amount of morpholine was seen. The lowest level tested corresponded to 7 mg m-3 (1/10 threshold limit value) for a 15 min sampling period with a sampling rate of 20 ml min-1. Exposed NIT-coated XAD-2 tubes were stable at room temperature for at least 2 weeks.

1-Naphthylisothiocyanate↗
PubMed2001

Characterization of chemical interferences in the determination of unsaturated aldehydes using aromatic hydrazine reagents and liquid chromatography.

A systematic investigation on interferences in the determination of unsaturated aldehydes and ketones using the 2,4-dinitrophenylhydrazine (DNPH) method is described. Acrolein, crotonaldehyde, methacrolein and 1-buten-3-one are derivatized with DNPH in the presence of an acidic catalyst to form the respective hydrazones. The unstable hydrazones react with excess reagent to form adducts. These are identified by high-performance liquid chromatography (HPLC)-mass spectrometry and spectroscopic techniques after cryogenic fraction collection of the adducts. The quantification of the unsaturated carbonyls with the DNPH method remains difficult. N-Methyl-4-hydrazino-7-nitrobenzofurazan (MNBDH) was used as an alternative reagent for this purpose. As with DNPH, the formation of a side product is observed. In contrast to DNPH, the alteration of the pH immediately after sampling leads to only one reaction product, which is stable and storable in solution at 4 degrees C for 2 days.

Aldehydes↗
PubMed2001

Aldehyde dehydrogenase. Maintaining critical active site geometry at motif 8 in the class 3 enzyme.

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved 'motif' segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.

Aldehyde Dehydrogenase↗
PubMed2001

Molecular medicine: a primer for clinicians. Part XII: DNA microarrays and their application to clinical medicine.

Previous papers in our Molecular Medicine series have described how the many tools of the molecular biologist are being used to develop practical bedside applications of modern molecular biology. We have discussed molecular diagnostics, gene therapy, and applications in clinical genetics. In this paper we discuss DNA microarray technology which provides a genome-wide profile of gene expression. We then describe some current and potential clinical applications in the disease diagnosis, prognosis and treatment. Particularly exciting is the potential of DNA array technology to provide individualized treatment for a wide variety of clinical conditions.

Gene Expression↗
PubMed2001

Molecular medicine: a primer for clinicians. Part XIII: The human genome project and the practice of medicine.

Publication earlier this year of the sequence of the human genome marked the end of one era of modern biology and initiated the beginning of another. This paper in our ongoing Molecular Medicine series briefly summarizes the history of the Human Genome Project, describes some of the major features of the structure and organization of the human genome and discusses some of the ways knowledge of the complete human genome might be clinically applicable in the near future.

Human Genome Project↗
PubMed2000

Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase.

Among pyridine-nucleotide-dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic residue, Glu140, coordinates the adenine ribose 2' hydroxyl. In many NAD-dependent dehydrogenases a similarly placed carboxylate is thought to be responsible for exclusion of NADP. The corresponding residue in most (approximately 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD-specific enzymes. Site-directed mutagenesis was performed on this residue in rat class 3 ALDH. Our results indicate that this residue contributes to tighter binding of NAD in the native enzyme, but suggest that additional factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essential for binding both coenzymes, consistent with its conservation in nearly all ALDHs (> 98%).

Aldehyde Dehydrogenase↗
PubMed1999

The same xenobiotic response element is required for constitutive and inducible expression of the mammalian aldehyde dehydrogenase-3 gene.

The mammalian aldehyde dehydrogenase-3 gene (ALDH3) exhibits several aspects of tissue-specific expression. Certain normal tissues, such as the cornea, constitutively express ALDH3 at very high levels. Other tissues, such as normal liver, do not express ALDH3. In liver, ALDH3 is inducible by polycyclic aromatic hydrocarbon xenobiotics by an Ah-receptor (AhR)-mediated pathway in which a liganded AhR complexes with nuclear ARNT protein, and the complex binds to a xenobiotic response element (XRE) sequence located near -3.0 kb in the ALDH3 5' flanking region and initiates transcription. We used our recently developed rat corneal epithelium culture model (Boesch et al., J. Biol. Chem. 271, 5150-5157, 1996) to study the molecular basis of constitutive ALDH3 expression. Transient transfection assays of corneal epithelium using a battery of ALDH3 5' flanking region-CAT reporter gene constructs indicate that high constitutive ALDH3 expression involves the same cis-acting elements as xenobiotic-induced ALDH3 expression in liver. These elements include a strong basal promoter region and the XRE located near -3.0 kb. Western analysis confirms the presence of AhR and ARNT proteins in 3-methylcholanthrene-treated rat liver, as well as ARNT protein in rat corneal epithelium. No AhR protein is found in rat cornea. The -3.0-kb ALDH3 XRE region contains multiple overlapping transcription factor binding sequences, including consensus sites for AhR, ARNT, HNF1, HNF4, and C/ebp. Electrophoretic mobility shift assays (EMSAs) indicate that constitutive expression of ALDH3 in cornea involves binding of ARNT, HNF1, and HNF4 to the ALDH3-XRE in an Ah-receptor-independent, ARNT-requiring manner. Transient transfection of ALDH3-CAT reporter gene constructs possessing a mutation in either the ARNT- or HNF4-DNA binding sites of the XRE confirms the functional importance of these sequence motifs in constitutive ALDH3 expression.

Aldehyde Dehydrogenase↗
PubMed1999

Negative regulation of rat hepatic aldehyde dehydrogenase 3 by glucocorticoids.

The expression of the aldehyde dehydrogenase 3 gene is known to be controlled by multiple regulatory processes. In liver, inducible expression appears to be mediated by two AhRE sequences which allow regulation of this gene by xenobiotic compounds which are ligands for the Ah receptor (Takimoto et al., 1994; this work). Constitutive expression of ALDH3 in tissues such as the cornea also involves the -3,500 region which contains an AhRE (Boesch et al., 1996; Boesch et al, 1998). However, the constellation of transcription factors which appear to interact with the AhRE in constitutively expressing corneal cells does not include either the Ah receptor nor the prototypical ARNT protein (Boesch et al., 1998). For both inducible and constitutive ALDH3 expression the more distal 5' flanking region sequences appear to interact with more proximal regulatory elements. Of particular interest is the region near -1 kb which includes the GC (-930 to -910) and cAMP (-1057 to -991) responsive elements as well as the 2 NF1 sites (-916 to -815), all of which appear to act as negative modulators of ALDH3 expression. A second putative ALDH3 negative regulatory region lies even more distal than -3,500 bp. To date, this region has been little studied, but appears to be involved in regulating both inducible and constitutive ALDH3 expression. This region may also be responsible for some of the tissue-specificity of ALDH3 expression. With respect to the work described here, in both isolated hepatocytes and HepG2 cells, no consistent negative regulation by glucocorticoids was observed in the basal expression of ALDH3. This indicates that the mechanism of GC-mediated negative regulation involves direct interference with ALDH3 gene activation mediated by the Ah receptor. Our results suggest a complex interplay between multiple transcription factors, including the GC and Ah receptors, regulates the hepatic expression of the ALDH3 gene. Active recruitment of transcription factors needed for gene transactivation, amelioration of the actions of negative regulatory trans-acting factors or cis-acting elements and/or chromatin remodeling may be required for achieve proper regulation of the aldehyde dehydrogenase 3 gene.

Aldehyde Dehydrogenase↗
PubMed1999

Inhibition of class-3 aldehyde dehydrogenase and cell growth by restored lipid peroxidation in hepatoma cell lines.

Hepatoma cells have a below-normal content of polyunsaturated fatty acids; this reduces lipid peroxidation and the production of cytotoxic and cytostatic aldehydes within the cells. In proportion to the degree of deviation, hepatoma cells also show an increase in the activity of Class-3 aldehyde dehydrogenase, an enzyme important in the metabolism of lipid peroxidation products and also in that of several drugs. When hepatoma cells with different degrees of deviation were enriched with arachidonic acid and stimulated to peroxidize by ascorbate/iron sulphate, their growth rate was reduced in proportion to the quantity of aldehydes produced and to the activity of aldehyde dehydrogenase. Therefore, 7777 cells, less deviated and with low Class-3 aldehyde dehydrogenase activity, were more susceptible to lipid peroxidation products than JM2 cells. It is noteworthy that repeated treatments with prooxidant also caused a decrease in mRNA and activity of Class-3 aldehyde dehydrogenase, contributing to the decreased growth and viability. Thus, Class-3 aldehyde dehydrogenase could be considered relevant for the growth of hepatoma cells, since it defends them against cell growth inhibiting aldehydes derived from lipid peroxidation.

Aldehyde Dehydrogenase↗