SummaryActinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin. Production of A40926 is governed by the stringent response at the transcriptional level. In fact, addition of an amino acid pool prevented the transcription of dbv cluster genes involved in the A40926 biosynthesis and the antibiotic production in chemically defined media, and a thiostrepton-resistant relaxed mutant was severely impaired in its ability to produce the antibiotic. The derivative strain rif19 , highly resistant to rifampicin (minimal inhibitory concentration, MIC > 200 m m m m g ml), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 m m m m g ml). In this strain A40926 production started earlier than in the wild type, and reached higher final levels. Moreover, the antibiotic production was not subjected to the stringent control. Molecular analysis led to the identification of two distinct rpoB alleles, rpoB S and rpoB R , in both the wild type and the rif19 . rpoB R harboured the H426N missense which is responsible for rifampicin-resistance in bacteria, in addition to other nucleotide substitutions affecting the primary structure of the RNA polymerase b b b b -chain. Transcript analysis revealed that rpoB R was expressed at a very low level in the wild type strain during the pseudo-exponential growth phase, and that the amount of rpoB R mRNA increased during the transition to the stationary phase. In contrast, expression of rpoB R was constitutive in the rif19 . The results of mRNA half-life analysis did not support the hypothesis that post-transcriptional events are responsible for the different rpoB expression patterns in the two strains, suggesting a role of transcriptional mechanisms.

SREBPs (sterol-regulatory-element-binding proteins) are a family of transcription factors that modulate the expression of several enzymes implicated in endogenous cholesterol, fatty acid, triacylglycerol and phospholipid synthesis. In the present study, evidence for SREBP-1 regulation at the translational level is reported. Using several experimental approaches, we have demonstrated that the 5'-UTR (untranslated region) of the SREBP-1a mRNA contains an IRES (internal ribosome entry site). Transfection experiments with the SREBP-1a 5'-UTR inserted in a dicistronic reporter vector showed a remarkable increase in the downstream cistron translation, through a cap-independent mechanism. Insertion of the SREBP-1c 5'-UTR in the same vector also stimulated the translation of the downstream cistron, but the observed effect can be ascribed, at least in part, to a cryptic promoter activity. Cellular stress conditions, such as serum starvation, caused an increase in the level of SREBP-1 precursor and mature form in both Hep G2 and HeLa cells, despite the overall reduction in protein synthesis, whereas mRNA levels for SREBP-1 were unaffected by serum starvation. Transfection experiments carried out with a dicistronic construct demonstrated that the cap-dependent translation was affected more than IRES-mediated translation by serum starvation. The thapsigargin- and tunicamycin-induced UPR (unfolded protein response) also increased SREBP-1 expression in Hep G2 cells, through the cap-independent translation mediated by IRES. Overall, these findings indicate that the presence of IRES in the SREBP-1a 5'-UTR allows translation to be maintained under conditions that are inhibitory to cap-dependent translation.

Heme is an essential molecule in many biological processes, such as transport and storage of oxygen and electron transfer as well as a structural component of hemoproteins. Defects of heme biosynthesis in developing erythroblasts have profound medical implications, as represented by sideroblastic anemia. The synthesis of heme requires the uptake of glycine into the mitochondrial matrix where glycine is condensed with succinyl coenzyme A to yield ␦-aminolevulinic acid. Herein we describe the biochemical and molecular characterization of yeast Hem25p and human SLC25A38, providing evidence that they are mitochondrial carriers for glycine. In particular, the hem25⌬ mutant manifests a defect in the biosynthesis of ␦-aminolevulinic acid and displays reduced levels of downstream heme and mitochondrial cytochromes. The observed defects are rescued by complementation with yeast HEM25 or human SLC25A38 genes. Our results identify new proteins in the heme biosynthetic pathway and demonstrate that Hem25p and its human orthologue SLC25A38 are the main mitochondrial glycine transporters required for heme synthesis, providing definitive evidence of their previously proposed glycine transport function. Furthermore, our work may suggest new therapeutic approaches for the treatment of congenital sideroblastic anemia.

The thyroid hormones (THs) 3,3′,5,5′-tetraiodo-l-thyronine (T4) and 3,5,3′-triiodo-l-thyronine (T3) influence many metabolic pathways. The major physiological function of THs is to sustain basal energy expenditure, by acting primarily on carbohydrate and lipid catabolism. Beyond the mobilization and degradation of lipids, at the hepatic level THs stimulate the de novo fatty acid synthesis (de novo lipogenesis, DNL), through both the modulation of gene expression and the rapid activation of cell signalling pathways. 3,5-Diiodo-l-thyronine (T2), previously considered only a T3 catabolite, has been shown to mimic some of T3 effects on lipid catabolism. However, T2 action is more rapid than that of T3, and seems to be independent of protein synthesis. An inhibitory effect on DNL has been documented for T2. Here, we give an overview of the mechanisms of THs action on liver fatty acid metabolism, focusing on the different effects exerted by T2 and T3 on the regulation of the DNL. The inhibitory action on DNL exerted by T2 makes this compound a potential and attractive drug for the treatment of some metabolic diseases and cancer.

Mitochondria are fundamental organelles producing energy and reactive oxygen species (ROS); their impaired functions play a key role in endothelial dysfunction. Hydroxytyrosol (HT), a well-known olive oil antioxidant, exerts health benefits against vascular diseases by improving endothelial function. However, the HT role in mitochondrial oxidative stress in endothelial dysfunction is not clear yet. To investigate the HT effects on mitochondrial ROS production in the inflamed endothelium, we used an in vitro model of endothelial dysfunction represented by cultured endothelial cells, challenged with phorbol myristate acetate (PMA), an inflammatory, prooxidant, and proangiogenic agent. We found that the pretreatment of endothelial cells with HT (1–30 μmol/L) suppressed inflammatory angiogenesis, a crucial aspect of endothelial dysfunction. The HT inhibitory effect is related to reduced mitochondrial superoxide production and lipid peroxidation and to increased superoxide dismutase activity. HT, in a concentration-dependent manner, improved endothelial mitochondrial function by reverting the PMA-induced reduction of mitochondrial membrane potential, ATP synthesis, and ATP5β expression. In PMA-challenged endothelial cells, HT also promoted mitochondrial biogenesis through increased mitochondrial DNA content and expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, nuclear respiratory factor-1, and mitochondrial transcription factor A. These results highlight that HT blunts endothelial dysfunction and pathological angiogenesis by ameliorating mitochondrial function, thus suggesting HT as a potential mitochondria-targeting antioxidant in the inflamed endothelium.

Bone regeneration is a complex process regulated by several factors that control overlapping biological processes, coordinating interactions among distinct cell populations. There is a great interest in identifying new strategies for inducing osteogenesis in a safe and efficient manner. Concentrated Growth Factor (CGF) is an autologous blood derived product obtained by centrifugation of venous blood following the procedure set on the Silfradent device. In this study the effects of CGF on osteogenic differentiation of human Bone Marrow Stem Cells (hBMSC) in vitro have been investigated; hBMSC were cultured with CGF or osteogenic medium, for 21 days. The osteogenic differentiation was evaluated measuring alkaline phosphatase (ALP) enzyme activity, matrix mineralization by alizarin red staining and through mRNA and protein quantification of osteogenic differentiation markers by Real-time PCR and Western blotting, respectively. The treatment with CGF stimulated ALP activity and promoted matrix mineralization compared to control and seems to be more effective than osteogenic medium. Also, hBMSC lost mesenchymal markers and showed other osteogenic features. Our study showed for the first time that CGF alone is able to induce osteogenic differentiation in hBMSC. The application of CGF on hBMSC osteoinduction might offer new clinical and biotechnological strategies in the tissue regeneration field.

The tricarboxylate carrier (TCC), an integral protein of the mitochondrial inner membrane, transports mitochondrial acetyl-CoA into the cytosol, where lipogenesis occurs. We investigated in rat liver mitochondria the effect of diets enriched with saturated fatty acids (beef tallow, BT), monounsaturated fatty acids (olive oil, OO) or n À 3 polyunsaturated fatty acids (fish oil, FO), respectively, on the activity and expression of TCC. TCC activity decreased, in parallel with TCC mRNA abundance, only upon FO-feeding. The TCC transcription rate, mRNA turnover and RNA processing indicated that FO administration regulates TCC gene at transcriptional and post-transcriptional steps, whereas BT-and OO-feeding do not seem to affect either TCC activity or gene expression.

Liver is an important target for thyroid hormone actions. T(3) exerts its effects by two mechanisms: (i) Genomic actions consisting of T(3) link to nuclear receptors that bind responsive elements in the promoter of target genes, (ii) non-genomic actions including integrin αvb3 receptor-mediated MAPK/ERK and PI3K/Akt/mTOR-C1 activation. SREBP-1a, SREBP-1c, and SREBP-2 are transcription factors involved in the regulation of lipogenic genes. We show in Hep G2 cells that T(3) determined a dose- and time-dependent increase in the level of the precursor form of SREBP-1 without affecting SREBP-1 mRNA abundance. T(3) also induced phosphorylation of ERK1/2, Akt and of mTOR-C1 target S6K-P70, and the cytosol-to-membrane translocation of PKC-α. Modulation of SREBP-1 protein level by T(3) was dependent on MAPK/ERK, PI3K/Akt/mTOR-C1 pathway activation since the MEK inhibitor PD98059 or the PI3K inhibitor LY294002 abolished the stimulatory effect of T(3) . Conversely, the effect of T(3) on SREBP-1 level was enhanced by using rapamycin, mTOR-C1 inhibitor. These data suggest a negative control of mTOR-C1 target S6K-P70 on PI3K/Akt pathway. The effect of T(3) on SREBP-1 content increased also by using PKC inhibitors. These inhibitors increased the action of T(3) on Akt phosphorylation suggesting that conventional PKCs may work as negative regulators of the T(3) -dependent SREBP-1 increase. T(3) effects were partially abrogated by tetrac, an inhibitor of the T(3) -αvβ3 receptor interaction and partially evoked by T(3) analog T(3) -agarose. These findings support a model in which T(3) activates intracellular signaling pathways which may be involved in the increment of SREBP-1 level through an IRES-mediated translation mechanism.