Saccharum spontaneum L. ‘SES 208’ genetic linkage map combining RFLP- and PCR-based markers

A 527 marker linkage map ofSaccharum spontaneum L. SES 208 (2n = 64) was established by analyzing 208 single-dose (SD) arbitrarily primed PCR polymorphisms, 234 SD RFLPs, 41 double-dose (DD) and one triple-dose (TD) polymorphisms. A map hypothesis constructed using these markers (minimum LOD = 4.00, = 0.25 M) had 64 linkage groups with 13 SD, nine DD, and one TD markers unlinked. Eight chromosome homology groups were identified by using DD fragments as well as SD RFLPs that identified more than one linkage group. Linkages in repulsion phase were absent from the map, as found in two previous genetic studies of this species. Together, these data demonstrate that SES 208 displayed polysomic segregation, a genetic behavior typical of autopolyploid species. As with previous studies, it was concluded that SES 208 behaved like an auto-octoploid, which was also in agreement with the number of homology groups observed. A ² was used to test whether the 527 markers were randomly distributed throughout the genome: both arbitrarily primed PCR markers and RFLPs had a distribution that was statistically indistinguishable from random. The integrated arbitrarily primed PCR-RFLP map had a predicted genomic coverage of 93% (considering only 442 SD polymorphisms) and an average interval between markers of 6 cM. SD markers were used to estimate the genome size of SES 208 at ca. 33 00 cM.     

Expression, Purification, and Characterization of Recombinant Human Factor X

A system is described for producing recombinant factor X with properties very similar to human plasma factor X. Optimization of the expression system for factor X resulted in the finding that human kidney cells (293 cells) are superior to the widely utilized baby hamster kidney cells (BHK cells) for the expression of functional factor X. It was also determined that production of factor X by 293 cells requires the substitution of the −2 residue (Thr → Arg) which affords the removal of the factor X propeptide. Purification of recombinant and plasma factor X is accomplished using a calcium-dependent monoclonal antibody directed against the gla domain. The proteins are comparable by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The rate and extent of activation by the factor X coagulant protein from Russell's viper venom and by factors IXa and VIIIa are similar; activation of the recombinant protein by VIIa and tissue factor is mildly faster. The activated enzymes have the same activity toward a chromogenic substrate and the biologic substrate, prothrombin. Both enzymes have the same apparent affinity for the activated platelet surface as judged by their ability to activate prothrombin. Finally, inhibition by antithrombin, with or without heparin, and inhibition by the tissue factor pathway inhibitor are equivalent. Recombinant factor X produced by this method is therefore well suited for probing structure–function relationships by mutational analysis.     

Isolation, Mapping, and Functional Expression of the Mouse X Chromosome Glycerol Kinase Gene

Glycerol kinase (Gyk) participates in the metabolism of endogenously derived and dietary glycerol. Deficiency of the human enzyme activity is an X-linked recessive disorder with a clinical picture varying from childhood metabolic crisis to asymptomatic adults incidentally identified by hyperlipidemia screening (pseudohypertriglyceridemia). Gyk is a member of a small group of kinases termed ambiquitous enzymes that are found in the cytosol or as membrane-bound enzymes associated with the voltage-dependent anion channel of the mitochondrial outer membrane. It was recently reported that in humans there are X-linked and autosomal copies of Gyk sequences, both apparently functional genes and processed pseudogenes. To understand the role of Gyk in normal metabolism and the variable clinical features seen with Gyk deficiency, we have characterized the mouse Gyk gene. We present the sequence of a full-length mouse Gyk cDNA that is alternatively spliced in brain. The Gyk gene was mapped to the mouse X chromosome by both fluorescencein situhybridization and an interspecies backcross panel, demonstrating conservation of synteny withdmd.To confirm the functional identity of the cDNA, transient transfection of the cDNA into COS7 cells was shown to cause a marked elevation in glycerol kinase activity.     

Pathways of glycosphingolipid biosynthesis in SW13 cells in the presence and absence of vimentin intermediate filaments

We reported previously that the incorporation of sugars intoglycosphingolipids (GSL) is diminished in SW13 cells that lacka vimentin intermediate filament (IF) network (vim–) comparedto vim+ cells. To further analyze the nature of this abnormality,we double-labeled cells with ³H-serine and ^l4C-sugars. Therewas no difference between vim+ and vim– cells in the incorporationof serine into GSL, although the usual difference in sugar incorporationwas observed. This indicated that the defect in vim– cellswas not in the incorporation of sugars into ceramide synthesizedde novo by acylation of sphinganine (pathway 1). Sugars canalso be incorporated into ceramide synthesized from sphingosinethat is derived from catabolism of sphin-golipids (pathway 2),and into GSL that recycle through the Golgi apparatus from endosomes(pathway 3). The amount of galactose and glucosamine incorporatedinto GSL in these three pathways was analyzed by the use oftwo inhibitors of sphingolipid biosynthesis. ß-Chloroala-nineinhibits the de novo synthesis of sphinganine (pathway 1), andfumonisin Bl inhibits the acylation of sphinganine and sphingosine(pathways 1 and 2). We were surprised to observe that in bothvim+ and vim– cells only 20–40% of sugar incorporationinto GSL took place in pathway 1, and 60–80% of sugarincorporation took place in the recycling pathways. Moreover,in contrast to larger GSL, GlcCer was not synthesized in pathway3. Our observations indicate that vimentin IF facilitate therecycling of GSL and sphingosine, and that the differences betweenvim+ and vim– cells are predominantly in pathways 2 and3. Furthermore, although it is generally believed that virtuallyall GSL are synthesized in the de novo pathway, these data indicatethat the recyling pathways predominate in the incorporationof sugars into GSL in SW13 cells. glycosphingolipid biosynthesis intermediate filaments SW13 cells sphingolipid recycling vimentin     

Novel plasma biomarker of atenolol-induced hyperglycemia identified through a metabolomics-genomics integrative approach

Introduction

While atenolol is an effective antihypertensive agent, its use is also associated with adverse events including hyperglycemia and incident diabetes that may offset the benefits of blood pressure lowering. By combining metabolomic and genomic data acquired from hypertensive individuals treated with atenolol, it may be possible to better understand the pathways that most impact the development of an adverse glycemic state.

Objective

To identify biomarkers that can help predict susceptibility to blood glucose excursions during exposure to atenolol.

Methods

Plasma samples acquired from 234 Caucasian participants treated with atenolol in the Pharmacogenomic Evaluation of Antihypertensive Responses trial were analyzed by gas chromatography Time-Of-Flight Mass Spectroscopy. Metabolomics and genomics data were integrated by first correlating participant’s metabolomic profiles to change in glucose after treatment with atenolol, and then incorporating genotype information from genes involved in metabolite pathways associated with glucose response.

Results

Our findings indicate that the baseline level of β-alanine was associated with glucose change after treatment with atenolol (Q = 0.007, β = 2.97 mg/dL). Analysis of genomic data revealed that carriers of the G allele for SNP rs2669429 in gene DPYS, which codes for dihydropyrimidinase, an enzyme involved in β-alanine formation, had significantly higher glucose levels after treatment with atenolol when compared with non-carriers (Q = 0.05, β = 2.76 mg/dL). This finding was replicated in participants who received atenolol as an add-on therapy (P = 0.04, β = 1.86 mg/dL).

Conclusion

These results suggest that β-alanine and rs2669429 may be predictors of atenolol-induced hyperglycemia in Caucasian individuals and further investigation is warranted.

     

Activation of β-Adrenergic Receptors Stimulates Release of an Inhibitory Transmitter from Astrocytes

Activation of beta-adrenergic receptors on astrocytes in primary cell culture results in the release of taurine, an inhibitory transmitter. Taurine release occurs via a cyclic AMP-mediated intracellular pathway, because (a) taurine release and intracellular cyclic AMP accumulation have similar pharmacologies and time courses of activation and (b) N6,O2'-dibutyryl cyclic AMP stimulates release with a time course similar to that observed with the beta-adrenergic agonist isoproterenol. These results describe a previously unrecognized physiological function of astrocytes in the CNS-receptor-mediated release of the neuroactive amino acid taurine. This observation indicates that astrocytes may function as local regulators of neuronal activity.     

Sorbitol production by Zymomonas mobilis

Summary High resolution ¹³C Nuclear Magnetic Resonance (NMR) spectroscopy has been employed to determine the chemical composition of the unknown major products in a sucrose or fructose plus glucose fermentation to ethanol by the bacterium Zymmonas mobilis. When grown on these sugars Z.mobilis was found to produce significant amounts of sorbitol, up to 43 g·l^-1 for strain ZM31 when grown on 250 g·l^-1 sucrose.The production of sorbitol and decrease of glucose, fructose, or sucrose was followed throughout batch fermentations by NMR and HPLC. Sorbitol was shown to be derived only from fructose by [¹⁴C]-feeding experiments. Additionally ³¹P NMR spectroscopy was utilized to determine the concentrations of both glucose 6-phosphate and fructose 6-phosphate relative to their respective concentrations in Z.mobilis cells fermenting glucose or fructose alone.It is suggested that free glucose inside the cell inhibits fructokinase. Free intracellular fructose may then be reduced to sorbitol via a dehydrogenase type enzyme. Attempts to grow Z.mobilis on sorbitol were unsuccessful, as were experiments to induce growth via mutagenesis.This work was supported in part by the National Energy Research, Development and Demonstration Council of Australia