Selective photodestruction of α-amino acids

A problem typically encountered in the analysis of amino acids in chemical evolution experiments and in extracts of meteorites is the large number present. For example, α-, β-, and γ-amino acids, N-mono substituted α-amino acids, and dicarboxylic α-amino acids have been found in extracts of the Murchison meteorite, and many more amino acids are present than have been positively identified by computerized gas chromatographic mass spectrometry. This paper reports an analytical method to selectively destroy the α-amino acids, with only the β- and γ-amino acids remaining in the solution. It is based on the ability of Cu²⁺ to complex with amino acids, the order of stability of these complexes being α > β > γ, = δ, = ε = 0. Aqueous solutions of α-amino acid-Cu²⁺ chelates are known to be decomposed by 254 nm light as well as by nonmonochromatic uv light, yielding a precipitate of Cu₂O. This paper shows that at 254 nm (ligand-metal charge transfer band) the rate of destruction of amino acids in Cu²⁺ aqueous solutions is in the following order, dicarboxylic α-amino acids > α-amino acids > N-monosubstituted α-amino acids β-amino acids ≈ γ-amino acids. Thus by irradiation with 254 nm light in the presence of Cu²⁺ all the amino acids can be destroyed except the β- and γ-amino acids. When almost 100% of the α-amino acids are destroyed, 80% of the β- and γ-amino acids still exist in solution. With this procedure, complex mixtures of amino acids can be simplified to make identification by gas chromatographic mass spectrometry casier.     

Study of hepatocyte differentiation using embryonic stem cells

The liver has many crucial functions including metabolizing dietary molecules, detoxifying compounds, and storing glycogen. The hepatocytes, comprising most of the liver organ, progressively modify their gene expression profile during the fetal development according to their roles in the different phases of development. Embryonic stem (ES) cells serve as a major tool in understanding liver development. These cells may also serve as a source of hepatic cells for cellular therapy. In this review, we aim to summarize the research that has been performed in the field of hepatocyte differentiation from mouse and human ES cells. We discuss the various methodologies for the differentiation of ES cells towards hepatic cells using either spontaneous or directed differentiation protocols. Although many protocols for differentiating ES cells to hepatic cells have been developed, the analysis of their status is not trivial and can lead to various conclusions. Hence, we discuss the issues of analyzing hepatocytes by means of the specificity of the markers for hepatocytes and the status of the cells as fetal or adult hepatocytes.     

The Hedgehog-inducible ubiquitin ligase subunit WSB-1 modulates thyroid hormone activation and PTHrP secretion in the developing growth plate

WSB-1 is a SOCS-box-containing WD-40 protein of unknown function that is induced by Hedgehog signalling in embryonic structures during chicken development. Here we show that WSB-1 is part of an E3 ubiquitin ligase for the thyroid-hormone-activating type 2 iodothyronine deiodinase (D2). The WD-40 propeller of WSB-1 recognizes an 18-amino-acid loop in D2 that confers metabolic instability, whereas the SOCS-box domain mediates its interaction with a ubiquitinating catalytic core complex, modelled as Elongin BC-Cul5-Rbx1 (ECS(WSB-1)). In the developing tibial growth plate, Hedgehog-stimulated D2 ubiquitination via ECS(WSB-1) induces parathyroid hormone-related peptide (PTHrP), thereby regulating chondrocyte differentiation. Thus, ECS(WSB-1) mediates a mechanism by which 'systemic' thyroid hormone can effect local control of the Hedgehog-PTHrP negative feedback loop and thus skeletogenesis.     

Interferon-gamma engages the p70 S6 kinase to regulate phosphorylation of the 40S S6 ribosomal protein

The signals generated by the IFNgamma receptor to initiate mRNA translation and generation of protein products that mediate IFNgamma responses are largely unknown. In the present study, we provide evidence for the existence of an IFNgamma-dependent signaling cascade activated downstream of the phosphatidylinositol (PI) 3'-kinase, involving the mammalian target of rapamycin (mTOR) and the p70 S6 kinase. Our data demonstrate that p70 S6K is rapidly phosphorylated and activated during engagement of the IFNgamma receptor in sensitive cell lines. Such activation of p70 S6 kinase is blocked by pharmacological inhibitors of the PI 3' kinase and mTOR, and is abrogated in double-knockout mouse embryonic fibroblasts for the alpha and beta isoforms of the p85 regulatory subunit of the PI 3'-kinase. The IFNgamma-activated p70 S6 kinase subsequently phosphorylates the 40S S6 ribosomal protein on serines 235/236, to regulate IFNgamma-dependent mRNA translation. In addition to phosphorylation of 40S ribosomal protein, IFNgamma also induces phosphorylation of the 4E-BP1 repressor of mRNA translation on threonines 37/46, threonine 70, and serine 65, sites whose phosphorylation is required for the inactivation of 4E-BP1 and its dissociation from the eukaryotic initiation factor-4E (eIF4E) complex. Thus, engagement of the PI 3'-kinase and mTOR by the IFNgamma receptor results in the generation of two distinct signals that play roles in the initiation of mRNA translation, suggesting an important role for this pathway in IFNgamma signaling.     

Utilization of [15N]glutamate by cultured astrocytes.

The metabolism of 0.25 mM-[15N]glutamic acid in cultured astrocytes was studied with gas chromatography-mass spectrometry. Almost all 15N was found as [2-15N]glutamine, [2-15N]glutamine, [5-15N]glutamine and [15N]alanine after 210 min of incubation. Some incorporation of 15N into aspartate and the 6-amino position of the adenine nucleotides also was observed, the latter reflecting activity of the purine nucleotide cycle. After the addition of [15N]glutamate the ammonia concentration in the medium declined, but the intracellular ATP concentration was unchanged despite concomitant ATP consumption in the glutamine synthetase reaction. Some potential sources of glutamate nitrogen were identified by incubating the astrocytes for 24 h with [5-15N]glutamine, [2-15N]glutamine or [15N]alanine. Significant labelling of glutamate was noted with addition of glutamine labelled on either the amino or the amide moiety, reflecting both glutaminase activity and reductive amination of 2-oxoglutarate in the glutamate dehydrogenase reaction. Alanine nitrogen also is an important source of glutamate nitrogen in this system.     

Effect of 5-amino-4-imidazolecarboxamide riboside on renal ammoniagenesis. Study with [15N]aspartate

[15N]Aspartate and 5-amino-4-imidazolecarboxamide riboside (AICAriboside) were used to evaluate the contribution of the purine nucleotide cycle to ammonia production in renal tubules isolated from control and chronically acidotic rats. Addition of 1 mM AICAriboside to incubation medium containing 2.5 mM [15N] aspartate significantly stimulated production of 15NH3 and 15N in the 6-amino group of adenine nucleotides during a 30-min incubation. In tubules from both control and acidotic animals, the levels of ATP, AMP, and NH3 were increased. In contrast, 5 mM AICAriboside inhibited 15NH3 production and reduced the total purine nucleotide content. In tubules from acidotic rats, enrichment in 15NH3 exceeded that in the 6-amino group of the adenine nucleotides, indicating that no precursor-product relationship existed between the purine nucleotide cycle and ammonia. Conversely, in tubules from control rats, 15N enrichment in the 6-amino group of the adenine nucleotides exceeded that in NH3. This relationship obtained whether or not AICAriboside was included in the incubation mixture. The current investigations show that the metabolism of aspartate through the purine nucleotide cycle is lower in renal tubules obtained from chronically acidotic rats than in control tubules. The observations indicate that AICAriboside has a biphasic effect on renal ammoniagenesis and adenine nucleotide synthesis, and suggest a possible clinical use of AICAriboside in cases of impaired ammonia formation in renal failure.     

The determination of amino acid pool sizes and turnover rates by gas chromatographic mass spectrometric analysis of stable isotope enrichment

Gas chromatography mass spectrometry has been used to determine the 15N enrichment of plasma glycine of rabbits at various times following the intravenous administration of 15N-glycine. These data were used to prepare isotope enrichment time decay curves for eleven individual animals. The slopes and intercepts of least squares lines that describe the decay curves were considerably more accurately than those reported in similar studies employing radioactive tracers. Individual glycine pool sizes (13.8-37.4 micronmoles per 100 g body wt), turnovers rates (2.66-3.36 pools h-1) and flux (50.4-99.7 micronmoles h-1 per 100 g body wt) were estimated from these parameters in a group of animals and compared with the literature values. These results demonstrate that low risk non-radioactive stable isotopes can be substituted for radioactive tracers in studies of human amino acid metabolism, with considerable saving in time and without loss in accuracy, when gas chromatography mass spectrometry is used to determine plasma amino acid and stable isotope enrichment.     

Comparison of defined culture systems for feeder cell free propagation of human embryonic stem cells

There are many reports of defined culture systems for the propagation of human embryonic stem cells in the absence of feeder cell support, but no previous study has undertaken a multi-laboratory comparison of these diverse methodologies. In this study, five separate laboratories, each with experience in human embryonic stem cell culture, used a panel of ten embryonic stem cell lines (including WA09 as an index cell line common to all laboratories) to assess eight cell culture methods, with propagation in the presence of Knockout Serum Replacer, FGF-2, and mouse embryonic fibroblast feeder cell layers serving as a positive control. The cultures were assessed for up to ten passages for attachment, death, and differentiated morphology by phase contrast microscopy, for growth by serial cell counts, and for maintenance of stem cell surface marker expression by flow cytometry. Of the eight culture systems, only the control and those based on two commercial media, mTeSR1 and STEMPRO, supported maintenance of most cell lines for ten passages. Cultures grown in the remaining media failed before this point due to lack of attachment, cell death, or overt cell differentiation. Possible explanations for relative success of the commercial formulations in this study, and the lack of success with other formulations from academic groups compared to previously published results, include: the complex combination of growth factors present in the commercial preparations; improved development, manufacture, and quality control in the commercial products; differences in epigenetic adaptation to culture in vitro between different ES cell lines grown in different laboratories.