Glucose Metabolism and Oxygen Availability Govern Reactivation of the Latent Human Retrovirus HTLV-1.

The human retrovirus HTLV-1 causes a hematological malignancy or neuroinflammatory disease in ∼10% of infected individuals. HTLV-1 primarily infects CD4 T lymphocytes and persists as a provirus integrated in their genome. HTLV-1 appears transcriptionally latent in freshly isolated cells; however, the chronically active anti-HTLV-1 cytotoxic T cell response observed in infected individuals indicates frequent proviral expression in vivo.

Bioassay-guided isolation of cytotoxic flavonoids from aerial parts of Coronopus didymus.

Ethnopharmacological Relevance: Coronopus didymus Linn. (Brassicaceae) is a medicinal plant used traditionally as antipyretic, expectorant, to purify blood and for alleviating symptoms of pain, inflammations, malaria, wounds and cancer.

Aim Of The Study: The present study was designed to isolate and identify the cytotoxic compounds responsible for anticancer activity from this traditionally useful medicinal plant.

Expression of Idh1 in the Murine Subventricular Zone Stem Cell Niche Recapitulates Features of Early Gliomagenesis.

Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed Idh1 in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate.

Variability in the carbon isotope composition of individual amino acids in plant proteins from different sources: 1 Leaves.

The natural carbon isotope composition of individual amino acids from plant leaf proteins has been measured to establish potential sources of variability. The plant leaves studied, taken from a range of plant groups (forbs, trees, grasses, and freshwater aquatic plants), showed no significant influence of either season or environment (water and light availability) on their Δδ(13)C values. Plant groups did, however, differ in carbon isotope composition, although no consistent differences were identified at the species level.

A method for continuous and stable perfusion of tissue and single cell preparations with accurate concentrations of volatile anaesthetics.

Background: It is difficult to design a system to reliably deliver volatile anaesthetics such as halothane or isoflurane to in vitro preparations such as tissues or cells cultures: the very volatility of the drugs means that they can rapidly dissipate from even carefully-prepared solutions. Furthermore, many experiments require the control of other gases (such as oxygen or carbon dioxide) which requires constant perfusion.

New Method: We describe a constant perfusion system that is air-tight (i.

Investigating the contribution of the active site environment to the slow reaction of hypoxia-inducible factor prolyl hydroxylase domain 2 with oxygen.

The prolyl hydroxylase domain proteins (PHDs) catalyse the post-translational hydroxylation of the hypoxia-inducible factor (HIF), a modification that regulates the hypoxic response in humans. The PHDs are Fe(II)/2-oxoglutarate (2OG) oxygenases; their catalysis is proposed to provide a link between cellular HIF levels and changes in O2 availability. Transient kinetic studies have shown that purified PHD2 reacts slowly with O2 compared with some other studied 2OG oxygenases, a property which may be related to its hypoxia-sensing role.

Non-enzymatic chemistry enables 2-hydroxyglutarate-mediated activation of 2-oxoglutarate oxygenases.

Accumulation of (R)-2-hydroxyglutarate in cells results from mutations to isocitrate dehydrogenase that correlate with cancer. A recent study reports that (R)-, but not (S)-2-hydroxyglutarate, acts as a co-substrate for the hypoxia-inducible factor prolyl hydroxylases via enzyme-catalysed oxidation to 2-oxoglutarate. Here we investigate the mechanism of 2-hydroxyglutarate-enabled activation of 2-oxoglutarate oxygenases, including prolyl hydroxylase domain 2, the most important human prolyl hydroxylase isoform.

A cell-permeable ester derivative of the JmjC histone demethylase inhibitor IOX1.

The 2-oxoglutarate (2OG)-dependent Jumonji C domain (JmjC) family is the largest family of histone lysine demethylases. There is interest in developing small-molecule probes that modulate JmjC activity to investigate their biological roles. 5-Carboxy-8-hydroxyquinoline (IOX1) is the most potent broad-spectrum inhibitor of 2OG oxygenases, including the JmjC demethylases, reported to date; however, it suffers from low cell permeability.

Optimal translational termination requires C4 lysyl hydroxylation of eRF1.

Efficient stop codon recognition and peptidyl-tRNA hydrolysis are essential in order to terminate translational elongation and maintain protein sequence fidelity. Eukaryotic translational termination is mediated by a release factor complex that includes eukaryotic release factor 1 (eRF1) and eRF3. The N terminus of eRF1 contains highly conserved sequence motifs that couple stop codon recognition at the ribosomal A site to peptidyl-tRNA hydrolysis.

Comparing ion-pairing reagents and sample dissolution solvents for ion-pairing reversed-phase liquid chromatography/electrospray ionization mass spectrometry analysis of oligonucleotides.

Rationale: A sensitive and selective liquid chromatography/mass spectrometry (LC/MS) method is essential for quality control of synthetic oligonucleotides. However, researchers are still searching for improvements to ion-pairing reagents for ion-pairing reversed-phase LC/MS. This study performed a comprehensive comparison of six ion-pairing reagents to determine their performance as mobile phase modifiers for oligonucleotide LC/MS.

Oxygenase-catalyzed ribosome hydroxylation occurs in prokaryotes and humans.

The finding that oxygenase-catalyzed protein hydroxylation regulates animal transcription raises questions as to whether the translation machinery and prokaryotic proteins are analogously modified. Escherichia coli ycfD is a growth-regulating 2-oxoglutarate oxygenase catalyzing arginyl hydroxylation of the ribosomal protein Rpl16. Human ycfD homologs, Myc-induced nuclear antigen (MINA53) and NO66, are also linked to growth and catalyze histidyl hydroxylation of Rpl27a and Rpl8, respectively.

Investigations on the oxygen dependence of a 2-oxoglutarate histone demethylase.

Histone N(ϵ)-methyl lysine demethylases are important in epigenetic regulation. KDM4E (histone lysine demethylase 4E) is a representative member of the large Fe(II)/2-oxoglutarate- dependent family of human histone demethylases. In the present study we report kinetic studies on the reaction of KDM4E with O2.

Variation of bone collagen amino acid δ13C values in archaeological humans and fauna with different dietary regimes: developing frameworks of dietary discrimination.

We present bone collagen amino acid (AA) δ(13)C values for a range of archaeological samples representing four "benchmark" human diet groups (high marine protein consumers, high freshwater protein consumers, terrestrial C(3) consumers, and terrestrial C(4) consumers), a human population with an "unknown" diet, and ruminants. The aim is to establish an interpretive palaeodietary framework for bone collagen AA δ(13)C values, and to assess the extent to which AA δ(13)C values can provide additional dietary information to bulk collagen stable isotope analysis. Results are analyzed to determine the ability of those AAs for which we have a complete set, to discriminate between the diet groups.

Single amino acid radiocarbon dating of Upper Paleolithic modern humans.

Archaeological bones are usually dated by radiocarbon measurement of extracted collagen. However, low collagen content, contamination from the burial environment, or museum conservation work, such as addition of glues, preservatives, and fumigants to "protect" archaeological materials, have previously led to inaccurate dates. These inaccuracies in turn frustrate the development of archaeological chronologies and, in the Paleolithic, blur the dating of such key events as the dispersal of anatomically modern humans.

The 2-oxoglutarate-dependent oxygenase JMJD6 catalyses oxidation of lysine residues to give 5S-hydroxylysine residues.

Amino acid analyses reveal that JMJD6-catalysed hydroxylation of RNA-splicing regulatory protein fragments occurs to give hydroxylysine products with 5S stereochemistry. This contrasts with collagen lysyl hydroxylases, which give 5R-hydroxylated products. The work suggests that more than one subfamily of lysyl hydroxylases has evolved and illustrates the importance of stereochemical assignments in proteomic analyses.

Preparative HPLC separation of underivatized amino acids for isotopic analysis.

Single-compound analysis of stable or radio-isotopes has found application in a number of fields ranging from archaeology to forensics. Often, the most difficult part of these analyses is the development of a method for isolating the compounds of interest.Here, we describe three complementary preparative HPLC procedures suitable for separating and isolating single amino acids from bone collagen or hair keratin with minimal isotopic contamination.

Review: Current applications and challenges for liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS).

High-precision isotope analysis is recognized as an essential research tool in many fields of study. Until recently, continuous flow isotope ratio mass spectrometry (CF-IRMS) was available via an elemental analyzer or a gas chromatography inlet system for compound-specific analysis of light stable isotopes. In 2004, however, an interface that coupled liquid chromatography with IRMS (LC/IRMS) became commercially available for the first time.

Liquid chromatography/isotope ratio mass spectrometry measurement of δ13C of amino acids in plant proteins.

In archaeological studies, the isotopic enrichment values of carbon and nitrogen in bone collagen give a degree of information on dietary composition. The isotopic enrichments of individual amino acids from bone collagen and dietary protein have the potential to provide more precise information about the components of diet. A limited amount of work has been done on this, although the reliability of these studies is potentially limited by fractionation arising through hydrolysis of whole plant tissue (where reaction between amino acids and carbohydrates may occur) and, for certain amino acids, the use of derivatives (particularly trifluoroacetyl derivatives) for gas chromatography/isotope ratio mass spectrometry (GC/IRMS) analysis.

Analysis of oligonucleotides by hydrophilic interaction liquid chromatography coupled to negative ion electrospray ionization mass spectrometry.

Hydrophilic interaction liquid chromatography (HILIC) is here successfully coupled to negative-ion electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS) for the analysis of synthetic and chemically modified oligonucleotides. Separation was performed on a 2.1 mm × 100 mm PEEK ZIC HILIC column packed with hydrophilic stationary phase with a permanent zwitterionic functional group and a particle size of 3.

The effect of dietary amino acid abundance and isotopic composition on the growth rate, metabolism and tissue δ13C of rainbow trout.

The aim of the present study was to test whether the dietary non-essential/conditionally essential amino acid composition has an effect on growth and protein utilisation and on δ13C of individual amino acids in rainbow trout (Oncorhynchus mykiss). Trout were reared on six purified diets containing only synthetic amino acids in place of protein. Diet 1 mimicked the amino acid composition of fishmeal, in diet 2, cysteine (Cys), glycine (Gly), proline (Pro) and tyrosine (Tyr) were isonitrogenously replaced by their precursor amino acids serine (Ser), glutamic acid (Glu) and phenylalanine (Phe), and in diet 3, alanine (Ala), asparagine and aspartate, Cys, Gly, Pro, Ser and Tyr were isonitrogenously replaced by Glu.

Biophysical characterisation of fibulin-5 proteins associated with disease.

FBLN5 encodes fibulin-5, an extracellular matrix calcium-binding glycoprotein that is essential for elastic fibre formation. FBLN5 mutations are associated with two distinct human diseases, age-related macular degeneration (AMD) and cutis laxa (CL), but the biochemical basis for the pathogenic effects of these mutations is poorly understood. Two missense mutations found in AMD patients (I169T and G267S) and two missense mutations found in CL patients (G202R and S227P) were analysed in a native-like context in recombinant fibulin-5 fragments.

Mixed-mode chromatography/isotope ratio mass spectrometry.

Liquid chromatography coupled to molecular mass spectrometry (LC/MS) has been a standard technique since the early 1970s but liquid chromatography coupled to high-precision isotope ratio mass spectrometry (LC/IRMS) has only been available commercially since 2004. This development has, for the first time, enabled natural abundance and low enrichment delta(13)C measurements to be applied to individual analytes in aqueous mixtures creating new opportunities for IRMS applications, particularly for the isotopic study of biological molecules. A growing number of applications have been published in a range of areas including amino acid metabolism, carbohydrates studies, quantification of cellular and plasma metabolites, dietary tracer and nucleic acid studies.

Amino acid delta13C analysis of hair proteins and bone collagen using liquid chromatography/isotope ratio mass spectrometry: paleodietary implications from intra-individual comparisons.

We report a novel method for the chromatographic separation and measurement of stable carbon isotope ratios (delta(13)C) of individual amino acids in hair proteins and bone collagen using the LC-IsoLink system, which interfaces liquid chromatography (LC) with isotope ratio mass spectrometry (IRMS). This paper provides baseline separation of 15 and 13 of the 18 amino acids in bone collagen and hair proteins, respectively. We also describe an approach to analysing small hair samples for compound-specific analysis of segmental hair sections.