Mammalian alcohol dehydrogenase — Functional and structural implications

Mammalian alcohol dehydrogenase (ADH) constitutes a complex system with different forms and extensive multiplicity (ADH1–ADH6) that catalyze the oxidation and reduction of a wide variety of alcohols and aldehydes. The ADH1 enzymes, the classical liver forms, are involved in several metabolic pathways beside the oxidation of ethanol, e.g. norepinephrine, dopamine, serotonin and bile acid metabolism. This class is also able to further oxidize aldehydes into the corresponding carboxylic acids, i.e. dismutation. ADH2, can be divided into two subgroups, one group consisting of the human enzyme together with a rabbit form and another consisting of the rodent forms. The rodent enzymes almost lack ethanol-oxidizing capacity in contrast to the human form, indicating that rodents are poor model systems for human ethanol metabolism. ADH3 (identical to glutathione-dependent formaldehyde dehydrogenase) is clearly the ancestral ADH form and S-hydroxymethylglutathione is the main physiological substrate, but the enzyme can still oxidize ethanol at high concentrations. ADH4 is solely extrahepatically expressed and is probably involved in first pass metabolism of ethanol beside its role in retinol metabolism. The higher classes, ADH5 and ADH6, have been poorly investigated and their substrate repertoire is unknown. The entire ADH system can be seen as a general detoxifying system for alcohols and aldehydes without generating toxic radicals in contrast to the cytochrome P450 system.     

Relationship of sequence and structure to specificity in the alpha-amylase family of enzymes

The hydrolases and transferases that constitute the alpha-amylase family are multidomain proteins, but each has a catalytic domain in the form of a (beta/alpha)(8)-barrel, with the active site being at the C-terminal end of the barrel beta-strands. Although the enzymes are believed to share the same catalytic acids and a common mechanism of action, they have been assigned to three separate families - 13, 70 and 77 - in the classification scheme for glycoside hydrolases and transferases that is based on amino acid sequence similarities. Each enzyme has one glutamic acid and two aspartic acid residues necessary for activity, while most enzymes of the family also contain two histidine residues critical for transition state stabilisation. These five residues occur in four short sequences conserved throughout the family, and within such sequences some key amino acid residues are related to enzyme specificity. A table is given showing motifs distinctive for each specificity as extracted from 316 sequences, which should aid in identifying the enzyme from primary structure information. Where appropriate, existing problems with identification of some enzymes of the family are pointed out. For enzymes of known three-dimensional structure, action is discussed in terms of molecular architecture. The sequence-specificity and structure-specificity relationships described may provide useful pointers for rational protein engineering.     

Neural stem cells in the adult human brain

New neurons are continuously generated in certain regions of the adult brain. Studies in rodents have shown that new neurons are generated from self-renewing multipotent neural stem cells. Here we demonstrate that both the lateral ventricle wall and the hippocampus of the adult human brain harbor self-renewing cells capable of generating neurons, astrocytes, and oligodendrocytes in vitro, i.e., bona fide neural stem cells.     

Peroxisome proliferator-induced long chain acyl-CoA thioesterases comprise a highly conserved novel multi-gene family involved in lipid metabolism

Long chain acyl-CoA esters are important intermediates in degradation and synthesis of fatty acids, as well as having important functions in regulation of intermediary metabolism and gene expression. Although the physiological functions for most acyl-CoA thioesterases have not yet been elucidated, previous data suggest that these enzymes may be involved in lipid metabolism by modulation of cellular concentrations of acyl-CoAs and fatty acids. In line with this, we have cloned four highly homologous acyl-CoA thioesterase genes from mouse, showing multiple compartmental localizations. The nomenclature for these genes has tentatively been assigned as CTE-I (cytosolic), MTE-I (mitochondrial), and PTE-Ia and Ib (peroxisomal), based on the identification of putative targeting signals. Although the various isoenzymes show between 67% and 94% identity at amino acid level, each individual enzyme shows a specific tissue expression. Our data suggest that all four genes are located within a very narrow cluster on chromosome 12 in mouse, similar to a sequence cluster on human chromosome 14, which identified four genes homologous to the mouse thioesterase genes. Four related genes were also identified in Caenorhabditis elegans, all containing putative PTS1 targeting signals, suggesting that the ancestral type I thioesterase gene(s) is/are of peroxisomal origin. All four thioesterases are differentially expressed in tissues examined, but all are inducible at mRNA level by treatment with the peroxisome proliferator clofibrate, or during the physiological condition of fasting, both of which conditions cause a perturbation in overall lipid homeostasis. These results strongly support the existence of a novel multi-gene family cluster of mouse acyl-CoA thioesterases, each with a distinct function in lipid metabolism.     

Social competition, corticosterone and survival in female lizard morphs

We examined the selective consequences of variation in behaviour and endocrine physiology in two female throat-colour morphs of the lizard, Uta stansburiana in the wild. Female morphs differed in home-range distribution patterns and corticosterone levels in relation to the density and frequency of their female neighbours. Levels of plasma corticosterone of yellow-throated females increased with increased density of both morphs. In contrast, orange-throated females had reduced levels of corticosterone in response to increased density of orange females. Additionally, females with lower corticosterone survived poorly, suggesting that social interactions and high local densities of orange females may be potentially costly for orange females. These results are consistent with decreased fitness effects and suppression of immune function previously reported for orange female morphs surrounded by more orange neighbours. These correlations, in conjunction with previous work in this system, indicate that corticosterone is likely to be an important physiological mechanism regulating female fitness in nature.     

Diving beetles (Dytiscidae) as predators of mosquito larvae (Culicidae) in field experiments and in laboratory tests of prey preference

Field experiments were performed in artificial ponds to evaluate how the density of predatory diving beetles (Dytiscidae) would affect the population levels of mosquito larvae (Culicidae). Mosquitoes colonizing the ponds were predominantly species of the genus Culex. In 2000, most of the dytiscids colonizing the ponds were small (Hydroporus spp.), and these predators had no impact on the size of larval mosquito populations, not even in ponds with added dytiscids. In 2001, larger beetles (Ilybius, Rhantus, and Agabus spp.) were more common, and there were significantly fewer mosquito larvae in ponds with the highest numbers of dytiscids. There was a negative correlation between numbers of diving beetles in the ponds and the mean body length of mosquito larvae. In neither year could dytiscid densities be maintained above a certain level owing to emigration. In laboratory tests, there were marked differences between three common dytiscid species in regard to preferences for Daphnia and Culex species as prey: Colymbetes paykulli Erichson chose mosquito larvae more often, whereas both Ilybius ater (De Geer) and I. fuliginosus (Fabricius) preferred Daphnia spp. All of the tested dytiscids consumed large numbers of prey. Since some dytiscid species can efficiently decrease populations of mosquito larvae, they are probably important in the natural control of these dipterans.     

Comparative enzymology of 11 beta -hydroxysteroid dehydrogenase type 1 from glucocorticoid resistant (Guinea pig) versus sensitive (human) species

Type 1 11 beta-hydroxysteroid dehydrogenase constitutes a prereceptor control mechanism through its ability to reduce dehydroglucocorticoids to the receptor ligands cortisol and corticosterone in vivo. We compared kinetic characteristics of the human and guinea pig 11 beta-hydroxysteroid dehydrogenase isozymes derived from species differing in glucocorticoid sensitivity. Both orthologs were successfully expressed as full-length enzymes in yeast and COS7 cells and as soluble transmembrane-deleted constructs in Escherichia coli. Both isozymes display Michaelis-Menten kinetics in intact cells and homogenates and show low apparent micromolar K(m) values in homogenates, which are lowered by approximately one order of magnitude in intact cells, allowing corticosteroid activation at physiological glucocorticoid levels. Recombinant soluble proteins were expressed and purified with high specific dehydrogenase and reductase activities, revealing several hundred-fold higher specificity constants than those reported earlier for the purified native enzyme. Importantly, these purified soluble enzymes also display a hyperbolic dependence of reaction velocity versus substrate concentration in 11-oxoreduction with K(m) values of 0.8 microm (human) and 0.6 microm (guinea pig), close to the values obtained from intact cells. Active site titration was carried out with the human enzyme using a novel inhibitor compound and reveals a fraction of 40-50% active sites/mol total enzyme. The kinetic data obtained argue against the involvement of 11 beta-hydroxysteroid dehydrogenase as a modulating factor for the glucocorticoid resistance observed in guinea pigs. Instead, the expression of 11 beta-hydroxysteroid dehydrogenase type 1 in the Zona glomerulosa of the guinea pig adrenal gland suggests a role of this enzyme in mineralocorticoid synthesis in this hypercortisolic species.     

Determination of laquinimod in plasma by coupled-column liquid chromatography with ultraviolet absorbance detection

Laquinimod is an immunomodulator that is currently in clinical trials. For pharmacokinetic and toxicokinetic studies in animals and humans a sensitive and accurate bioanalytical method was required. In this paper a bioanalytical method for the determination of laquinimod by liquid chromatography is described. After a protein precipitation step the plasma sample was injected onto a coupled-column HPLC system. After further purification from macromolecules on a short restricted access material C(18) column the analyte was transferred to a reversed-phase C(18) analytical column and separated from interfering substances. The analyte was detected by UV detection. The method was validated with respect to linearity, selectivity, precision, accuracy, limit of quantitation, limit of detection, recovery and stability. The limit of quantitation was 0.75 micromol/L, the intermediate precision was 1.8-3.6% (C.V.) and the accuracy was 97.7-114.7%. In conclusion, the method was found to perform well and is suitable for use in pharmacokinetic and toxicokinetic studies.     

Proteome analysis of grain filling and seed maturation in barley

In monocotyledonous plants, the process of seed development involves the deposition of reserves in the starchy endosperm and development of the embryo and aleurone layer. The final stages of seed development are accompanied by an increase in desiccation tolerance and drying out of the mature seed. We have used two-dimensional gel electrophoresis for a time-resolved study of the changes in proteins that occur during seed development in barley (Hordeum vulgare). About 1,000 low-salt extractable protein spots could be resolved on the two-dimensional gels. Protein spots were divided into six categories according to the timing of appearance or disappearance during the 5-week period of comparison. Nineteen different proteins or protein fragments in 36 selected spots were identified by matrix-assisted laser-desorption ionization time of flight mass spectrometry (MS) or nano-electrospray tandem MS/MS. Some proteins were present throughout development (for example, cytosolic malate dehydrogenase), whereas others were associated with the early grain filling (ascorbate peroxidase) or desiccation (Cor14b) stages. Most noticeably, the development process is characterized by an accumulation of low-M(r) alpha-amylase/trypsin inhibitors, serine protease inhibitors, and enzymes involved in protection against oxidative stress. We present examples of proteins not previously experimentally observed, differential extractability of thiol-bound proteins, and possible allele-specific spot variation. Our results both confirm and expand on knowledge gained from previous analyses of individual proteins involved in grain filling and maturation.     

A neuroproteomic approach to targeting neuropeptides in the brain

A novel universal neuropeptide display approach in the mass range of 300-5000 Da was developed to complement two-dimensional gel electrophoresis in the analysis of peptides and small proteins from brain tissue samples. For the analysis of neuropeptides we utilized on-line nanoscale capillary reversed phase liquid chromatography and electrospray ionization quadrupole-time of flight mass spectrometry. The method was employed for the analysis of a large number of peptides from three specific rat brain regions. Approximately 1500 peptides from each brain region were detected in the same analysis. Several of these peptides were sequenced using collision-induced dissociation and identified by database search tools. In addition, a method for comparing peptide elution profiles between samples was developed, to provide two- and three-dimensional computer graphics of the profiles and to pinpoint differences for statistical measurements. Among the characterized peptides were fragments from proteins such as hemoglobin, alpha-synuclein, stathmin, cyclophilin, actin, NADH dehydrogenase, cytochrome c oxidase and prosomatostatin, as well as the bioactive neuropeptides W-hemorphin-4, and LW-hemorphin-7. The present study showed that the combination of nanoscale reversed phase liquid chromatography and high-resolution tandem mass spectrometry provides a novel and powerful approach to investigate a large number of peptides and protein fragments in the brain.