Divergent β-hairpins determine double-strand versus single-strand substrate recognition of human AlkB-homologues 2 and 3

Human AlkB homologues ABH2 and ABH3 repair 1-methyladenine and 3-methylcytosine in DNA/RNA by oxidative demethylation. The enzymes have similar overall folds and active sites, but are functionally divergent. ABH2 efficiently demethylates both single- and double-stranded (ds) DNA, whereas ABH3 has a strong preference for single-stranded DNA and RNA. We find that divergent F1 β-hairpins in proximity of the active sites of ABH2 and ABH3 are central for substrate specificities. Swapping F1 hairpins between the enzymes resulted in hybrid proteins resembling the donor proteins. Surprisingly, mutation of the intercalating residue F102 had little effect on activity, while the double mutant V101A/F102A was catalytically impaired. These residues form part of an important hydrophobic network only present in ABH2. In this functionally important network, F124 stacks with the flipped out base while L157 apparently functions as a buffer stop to position the lesion in the catalytic pocket for repair. F1 in ABH3 contains charged and polar residues preventing use of dsDNA substrate. Thus, E123 in ABH3 corresponds to F102 in ABH2 and the E123F-variant gained capacity to repair dsDNA with no loss in single strand repair capacity. In conclusion, divergent sequences outside of the active site determine substrate specificities of ABH2 and ABH3

Jord- og vannovervåking i landbruket (JOVA). Feltrapporter fra programmet i 2006.

Program for Jord- og vannovervåking i landbruket (JOVA) ledes av Bioforsk Jord og miljø, og utføres i samarbeid med en rekke andre institusjoner. Programmet rapporterer årlig overvåkingsresultater fra jordbruksdominerte nedbørfelt over hele landet. Feltene representerer ulike driftsformer, jordbunnsforhold, og hydrologiske og klimatiske forhold. De årlige feltrapportene beskriver jordbruksdrift, og avrenning og tap av næringsstoffer og partikler i de ulike feltene. Tap av partikler og næringsstoffer rapporteres for agrohydrologisk år, 1. mai - 30. april, mens tap av pesticider rapporteres for kalenderår.publishedVersio

Jord- og vannovervåking i landbruket (JOVA). Timebekken 2006

Overvåkingen av Timebekken inngår som en del av programmet Jord- og vannovervåking i landbru-ket (JOVA) og har pågått siden 1992. Feltet overvåkes med hensyn på erosjon og næringsstoffavren-ning, og pesticider.publishedVersio

A Sterol Panel for Multiple Rare Lipid Disorders: Validation and Application for Sitosterolemia, Cerebrotendinous Xanthomatosis and Smith-Lemli-Opitz Syndome

BACKGROUND. Disease-specific sterols accumulate in the blood of patients with several rare lipid disorders. Biochemical measurement of these sterols is important for correct diagnosis and sometimes monitoration of treatment. Existing methods to measure sterols in blood, particularly plant sterols, are often laborious and time consuming. Partly as a result, clinical access to sterol measurements are limited in many parts of the world. METHODS. A simple and rapid method to extract free sterols from human serum and quantitate their concentration using isotope-dilution liquid chromatography tandem mass spectrometry (LC-MS/MS) without derivatization was developed. The method was designed to be “compatible” with routine workflows (eg. 96-well format) in a clinical lab and was extensively validated. Serum from 73 controls were analyzed and used to estimate the upper reference limits for sitosterol, campesterol, stigmasterol, desmosterol, 7-dehydrocholesterol (7DHC), lathosterol and cholestanol. Serum from patients with the rare lipid disorders sitosterolemia (n=7), Smith-Lemli-Optiz syndrome (SLOS; n=1) and cerebrotendinous xanthomatosis (CTX; n=1) were analyzed. RESULTS. All seven sitosterolemia patients were measured to have greatly elevated levels of free plant sterols (sitosterol, campesterol and stigmasterol) compared to the controls. The SLOS serum contained massively increased concentrations of 7DHC and an unidentified compound (likely 8-dehydrocholesterol). CTX serum contained greatly increased concentrations of cholestanol, as well as 7DHC and lathosterol. Spiking experiments indicated that the method is likely also useful in the diagnosis of desmosterolosis and lathosterolosis. CONCLUSION. The reported method is a relatively simple and fast method capable of quantitating diagnostically important sterols and differentiating patients with several rare lipid disorders from controls

Jord- og vannovervåking i landbruket (JOVA). Feltrapporter fra programmet i 2006.

Program for Jord- og vannovervåking i landbruket (JOVA) ledes av Bioforsk Jord og miljø, og utføres i samarbeid med en rekke andre institusjoner. Programmet rapporterer årlig overvåkingsresultater fra jordbruksdominerte nedbørfelt over hele landet. Feltene representerer ulike driftsformer, jordbunnsforhold, og hydrologiske og klimatiske forhold. De årlige feltrapportene beskriver jordbruksdrift, og avrenning og tap av næringsstoffer og partikler i de ulike feltene. Tap av partikler og næringsstoffer rapporteres for agrohydrologisk år, 1. mai - 30. april, mens tap av pesticider rapporteres for kalenderår.publishedVersio

Jord- og vannovervåking i landbruket (JOVA). Feltrapporter fra programmet i 2006.

Program for Jord- og vannovervåking i landbruket (JOVA) ledes av Bioforsk Jord og miljø, og utføres i samarbeid med en rekke andre institusjoner. Programmet rapporterer årlig overvåkingsresultater fra jordbruksdominerte nedbørfelt over hele landet. Feltene representerer ulike driftsformer, jordbunnsforhold, og hydrologiske og klimatiske forhold. De årlige feltrapportene beskriver jordbruksdrift, og avrenning og tap av næringsstoffer og partikler i de ulike feltene. Tap av partikler og næringsstoffer rapporteres for agrohydrologisk år, 1. mai - 30. april, mens tap av pesticider rapporteres for kalenderår.publishedVersio

Jord- og vannovervåking i landbruket (JOVA). Feltrapporter fra programmet i 2007

Program for Jord- og vannovervåking i landbruket (JOVA) ledes av Bioforsk Jord og miljø, og utføres i samarbeid med en rekke andre institusjoner. Programmet rapporterer årlig overvåkingsresultater fra jordbruksdominerte nedbørfelt over hele landet. Feltene representerer ulike driftsformer, jordbunnsforhold, og hydrologiske og klimatiske forhold. De årlige feltrapportene beskriver jordbruksdrift, og avrenning og tap av næringsstoffer og partikler i de ulike feltene. Tap av partikler og næringsstoffer rapporteres for agrohydrologisk år, 1. mai - 30. april, mens tap av pesticider rapporteres for kalenderår.publishedVersio

Human AlkB Homolog 1 Is a Mitochondrial Protein That Demethylates 3-Methylcytosine in DNA and RNA*

The Escherichia coli AlkB protein and human homologs hABH2 and hABH3 are 2-oxoglutarate (2OG)/Fe(II)-dependent DNA/RNA demethylases that repair 1-methyladenine and 3-methylcytosine residues. Surprisingly, hABH1, which displays the strongest homology to AlkB, failed to show repair activity in two independent studies. Here, we show that hABH1 is a mitochondrial protein, as demonstrated using fluorescent fusion protein expression, immunocytochemistry, and Western blot analysis. A fraction is apparently nuclear and this fraction increases strongly if the fluorescent tag is placed at the N-terminal end of the protein, thus interfering with mitochondrial targeting. Molecular modeling of hABH1 based upon the sequence and known structures of AlkB and hABH3 suggested an active site almost identical to these enzymes. hABH1 decarboxylates 2OG in the absence of a prime substrate, and the activity is stimulated by methylated nucleotides. Employing three different methods we demonstrate that hABH1 demethylates 3-methylcytosine in single-stranded DNA and RNA in vitro. Site-specific mutagenesis confirmed that the putative Fe(II) and 2OG binding residues are essential for activity. In conclusion, hABH1 is a functional mitochondrial AlkB homolog that repairs 3-methylcytosine in single-stranded DNA and RNA