A new high performance liquid chromatographic technique for separation and determination of adenylic and nicotinamide nucleotides in Lactobacillus plantarum

A new technique for separation and determination of ATP, ADP, AMP, NAD⁺, NADP⁺, NADH and NADPH in Lacwbacillus plantarum has been developped. It involves acid and basic extraction of nucleotide pool, analysis by reverse-phase high performance liquid chromatography on a 5 m Spherisorb ODS-1 column and UV detection. The method offers advantages in accurate estimations of adenylic and nicotinamide nucleotides concentrations in Lactobacillus plantarum during the growth phase. Such results indicate the potential of this technique as an important research tool.     

Purification and properties of a recombinant sulfur analog of murine selenium-glutathione peroxidase.

We previously constructed plasmids for synthesis of glutathione-peroxidase (GPx) mutants in an Escherichia coli expression system. In these recombinant proteins either cysteine ([Cys]GPx mutant) or serine ([Ser]GPx mutant) were present in place of the active-site selenocysteine (SeCys) of the natural enzyme. We have now investigated GPx activity of [Cys]GPx and [Ser]GPx mutants. Enzyme assays performed on preparations of these partially purified proteins demonstrated that the [Cys]GPx mutant exhibited a significant GPx activity, unlike the [Ser]GPx mutant. Purification of [Cys]GPx was performed in two steps of ion-exchange chromatography giving a 98% homogenous protein in 50% yield. The purified [Cys]GPx protein was shown to be a symmetrical tetramer by the means of gel-filtration HPLC and SDS/PAGE. Two isoelectric points were found (6.8 and 7.2) which may reflect two different oxidation states of the mutant protein. The GPx activity of the [Cys]GPx mutant was optimal at pH 8.5. The [Cys]GPx mutant had a specific activity approximately 1000-fold smaller than that of the natural enzyme, and was very easily inactivated by hydroperoxides. Inhibition of the activity with iodoacetate determined a pKa of 8.3, presumably that of the active-site cysteine. Unlike that of SeGPx, the GPx activity of [Cys]GPx was only slightly inhibited by mercaptosuccinate. We discuss hypothetical mechanistic constraints of either catalytic cycle, which may explain such results.     

Free and kerogen‐bound biomarkers from late Tonian sedimentary rocks record abundant eukaryotes in mid‐Neoproterozoic marine communities

Lipid biomarker assemblages preserved within the bitumen and kerogen phases of sedimentary rocks from the ca. 780–729 Ma Chuar and Visingsö Groups facilitate paleoenvironmental reconstructions and reveal fundamental aspects of emerging mid‐Neoproterozoic marine communities. The Chuar and Visingsö Groups were deposited offshore of two distinct paleocontinents (Laurentia and Baltica, respectively) during the Tonian Period, and the rock samples used had not undergone excessive metamorphism. The major polycyclic alkane biomarkers detected in the rock bitumens and kerogen hydropyrolysates consist of tricyclic terpanes, hopanes, methylhopanes, and steranes. Major features of the biomarker assemblages include detectable and significant contribution from eukaryotes, encompassing the first robust occurrences of kerogen‐bound regular steranes from Tonian rocks, including 21‐norcholestane, 27‐norcholestane, cholestane, ergostane, and cryostane, along with a novel unidentified C₃₀ sterane series from our least thermally mature Chuar Group samples. Appreciable values for the sterane/hopane (S/H) ratio are found for both the free and kerogen‐bound biomarker pools for both the Chuar Group rocks (S/H between 0.09 and 1.26) and the Visingsö Group samples (S/H between 0.03 and 0.37). The more organic‐rich rock samples generally yield higher S/H ratios than for organic‐lean substrates, which suggests a marine nutrient control on eukaryotic abundance relative to bacteria. A C₂₇ sterane (cholestane) predominance among total C₂₆–C₃₀ steranes is a common feature found for all samples investigated, with lower amounts of C₂₈ steranes (ergostane and crysotane) also present. No traces of known ancient C₃₀ sterane compounds; including 24‐isopropylcholestanes, 24‐n‐propylcholestanes, or 26‐methylstigmastanes, are detectable in any of these pre‐Sturtian rocks. These biomarker characteristics support the view that the Tonian Period was a key interval in the history of life on our planet since it marked the transition from a bacterially dominated marine biosphere to an ocean system which became progressively enriched with eukaryotes. The eukaryotic source organisms likely encompassed photosynthetic primary producers, marking a rise in red algae, and consumers in a revamped trophic structure predating the Sturtian glaciation.     

Cell-Type-Specific Gene Expression Profiling in Adult Mouse Brain Reveals Normal and Disease-State Signatures

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Inhibition of B56-containing protein phosphatase 2As by the early response gene IEX-1 leads to control of Akt activity

The importance of PP2A in the regulation of Akt/PKB activity has long been recognized but the nature of the holoenzyme involved and the mechanisms controlling dephosphorylation are not yet known. We identified IEX-1, an early gene product with proliferative and survival activities, as a specific inhibitor of B56 regulatory subunit-containing PP2A. IEX-1 inhibits B56-PP2A activity by allowing the phosphorylation of B56 by ERK. This leads to sustained ERK activation. IEX-1 has no effect on PP2A containing other B family subunits. Thus, studying IEX-1 contribution to signaling should help the discovery of new pathways controlled by B56-PP2A. By using overexpression and RNA interference, we show here that IEX-1 increases Akt/PKB activity in response to various growth factors by preventing Akt dephosphorylation on both Thr(308) and Ser(473) residues. PP2A-B56beta and gamma subunits have the opposite effect and reverse IEX-1-mediated Akt activation. The effect of IEX-1 on Akt is ERK-dependent. Indeed: (i) a IEX-1 mutant deficient in ERK binding had no effect on Akt; (ii) ERK dominant-negative mutants reduced IEX-1-mediated increase in pAkt; (iii) a B56beta mutant that cannot be phosphorylated in the ERK.IEX-1 complex showed an enhanced ability to compete with IEX-1. These results identify B56-containing PP2A holoenzymes as Akt phosphatases. They suggest that IEX-1 behaves as a general inhibitor of B56 activity, enabling the control of both ERK and Akt signaling downstream of ERK.     

Carrier-mediated uptake and phloem systemy of a 350-Dalton chlorinated xenobiotic with an alpha-amino acid function

In a previous paper we have shown that epsilon-(phenoxyalkanecarboxylyl)-L-Lys conjugates are potent inhibitors of amino acid transport systems and that it is possible to modulate the uptake inhibition by hydrophobic or hydrophilic additions in the 4-position of the aromatic ring (J.F. Chollet, C. Delétage, M. Faucher, L. Miginiac, J.L. Bonnemain [1997] Biochem Biophys Acta 1336: 331-341). In this report we demonstrate that epsilon-(2,4-dichlorophenoxyacetyl)-L-Lys (2,4D-Lys), one of the largest molecules of the series and one of the most potent inhibitors, is a highly permeant conjugate. Uptake of 2,4D-Lys by broad bean (Vicia faba) leaf discs is mediated by an active carrier system (Km1 = 0.2 mM; Vmax1 = 2.4 nmol x cm(-2) x h(-1) at pH 5.0) complemented by an important diffusive component. Among the compounds tested (neutral, basic, and acidic amino acids, auxin, glutathione, and sugars), only the aromatic amino acids clearly compete with 2,4D-Lys. The conjugate accumulates in the vein network, is exported toward the growing organs, and exhibits a distribution pattern different from that of the herbicide moiety. However, over time 2,4D-Lys progressively splits into 2,4D and lysine. Analyses by high-performance liquid chromatography and liquid scintillation spectrometry of the phloem sap collected from the castor bean system, used as a systemy test, indicate decreasing capacities of 2,4D, 2,4D-Lys, and glyphosate, respectively, to move from the epidermis cell wall to the sieve element. Our results show that it is possible to design synthesis of large-size xenobiotics (approximately 350 D) with a lipophilic pole, exhibiting high mobility within the vascular system.     

Effects of corticosterone on muscle mitochondria identifying different sensitivity to glucocorticoids in Lewis and Fischer rats

Previous studies in rat have demonstrated decreased number of mitochondria and uncoupling of oxidative phosphorylation after administration of glucocorticoids but at supraphysiological doses and using synthetic glucocorticoids. To analyze the relationships between corticosterone levels (the natural glucocorticoid in rat) and muscle mitochondrial metabolism, Lewis and Fischer 344 rats were bilaterally adrenalectomized and implanted with different corticosterone pellets (0, 12, 50, 100, and 200 mg of corticosterone). Rats bearing a corticosterone pellet delivering corticosterone at concentrations in the range of chronic stress-induced levels presented a lower amount of functional muscle mitochondria with a decrease in cytochrome c oxidase and citrate synthase activities and a depletion of mitochondrial DNA. Moreover, a strain difference in tissue sensitivity to corticosterone was depicted both in end-organ sensitive to glucocorticoids (body, thymus, and adrenal weights) and in muscle mitochondrial metabolism (Lewis > Fischer). Interestingly, this strain difference was also observed in the absence of corticosterone, with a deleterious effect on muscle mitochondrial metabolism in Fischer rats, whereas no effects were observed in Lewis rats. We therefore postulate that corticosterone is necessary for muscle mitochondrial metabolism exerting its effects in Fischer rats with an inverted U curve, whereby too little (only Fischer) or too much (Fischer and Lewis) corticosterone is deleterious to muscle mitochondrial metabolism. In conclusion, we propose a general model of coordinate regulation of mitochondrial energetic metabolism by glucocorticoids.