1H-NMR study of the quadruplex [d(TGGGT)]4 containing a modified thymine

A NMR structural study of quadruplex [d(TGGGT)]4 containing a modified thymine is reported. The three dimensional structure of the complex is very similar to those of other parallel stranded quadruplexes. The modified thymines (T*) are able, at least in the minimised structures, to form a tetrad containing extra H-bonds through the hydroxyl groups. Nevertheless, in this new tetrad the modified thymines are slightly open towards the solvent respect to the unmodified T-tetrad.     

Fatty acid transport in Saccharomyces cerevisiae. Directed mutagenesis of FAT1 distinguishes the biochemical activities associated with Fat1p

The fatty acid transport protein Fat1p functions as a component of the long-chain fatty acid transport apparatus in the yeast Saccharomyces cerevisiae. Fat1p has significant homologies to the mammalian fatty acid transport proteins (FATP) and the very long-chain acyl-CoA synthetases (VLACS). In order to further understand the functional roles intrinsic to Fat1p (fatty acid transport and VLACS activities), a series of 16 alleles carrying site-directed mutations within FAT1 were constructed and analyzed. Sites chosen for the construction of amino acid substitutions were based on conservation between Fat1p and the mammalian FATP orthologues and included the ATP/AMP and FATP/VLACS signature motifs. Centromeric and 2 mu plasmids encoding mutant forms of Fat1p were transformed into a yeast strain containing a deletion in FAT1 (fat1Delta). For selected subsets of FAT1 mutant alleles, we observed differences between the wild type and mutants in 1) growth rates when fatty acid synthase was inhibited with 45 microm cerulenin in the presence of 100 microm oleate (C(18:1)), 2) levels of fatty acid import monitored using the accumulation of the fluorescent fatty acid 4,4-difluoro-5-methyl-4-bora-3a,4a-diaza-S-indacene-3-dodecanoic acid and [(3)H]oleate, 3) levels of lignoceryl (C(24:0)) CoA synthetase activities, and 4) fatty acid profiles monitored using gas chromatography/mass spectrometry. In most cases, there was a correlation between growth on fatty acid/cerulenin plates, the levels of fatty acid accumulation, very long-chain fatty acyl-CoA synthetase activities, and the fatty acid profiles in the different FAT1 mutants. For several notable exceptions, the fatty acid transport and very long-chain fatty acyl-CoA synthetase activities were distinguishable. The characterization of these novel mutants provides a platform to more completely understand the role of Fat1p in the linkage between fatty acid import and activation to CoA thioesters.     

Mitogen-activated protein kinases and NF-kappa B are involved in TNF-alpha responses to group B streptococci

TNF-alpha is a mediator of lethality in experimental infections by group B streptococcus (GBS), an important human pathogen. Little is known of signal transduction pathways involved in GBS-induced TNF-alpha production. Here we investigate the role of mitogen-activated protein kinases (MAPKs) and NF-kappa B in TNF-alpha production by human monocytes stimulated with GBS or LPS, used as a positive control. Western blot analysis of cell lysates indicates that extracellular signal-regulated kinase 1/2 (ERK 1/2), p38, and c-Jun N-terminal kinase MAPKs, as well as I kappa B alpha, became phosphorylated, and hence activated, in both LPS- and GBS-stimulated monocytes. The kinetics of these phosphorylation events, as well as those of TNF-alpha production, were delayed by 30-60 min in GBS-stimulated, relative to LPS-stimulated, monocytes. Selective inhibitors of ERK 1/2 (PD98059 or U0126), p38 (SB203580), or NF-kappa B (caffeic acid phenetyl ester (CAPE)) could all significantly reduce TNF-alpha production, although none of the inhibitors used alone was able to completely prevent TNF-alpha release. However, this was completely blocked by combinations of the inhibitors, including PD98059-SB203580, PD98059-CAPE, or SB203580-CAPE combinations, in both LPS- and GBS-stimulated monocytes. In conclusion, our data indicate that the simultaneous activation of multiple pathways, including NF-kappa B, ERK 1/2, and p38 MAPKs, is required to induce maximal TNF-alpha production. Accordingly, in septic shock caused by either GBS or Gram-negative bacteria, complete inhibition of TNF-alpha release may require treatment with drugs or drug combinations capable of inhibiting multiple activation pathways.     

Attenuation of Excitatory Amino Acid Toxicity by Metabotropic Glutamate Receptor Agonists and Aniracetam in Primary Cultures of Cerebellar Granule Cells

Abstract: Activation of glutamate ionotropic receptors represents the primary event in the neurotoxicity process triggered by excitatory amino acids. We demonstrate here that the concentration-dependent stimulation of metabotropic glutamate receptor (mGluR) by the selective agonist trans-1-aminocyclopentane-1, 3-dicarboxylate or by quisqualate counteracts both glutamate- and kainate-induced neurotoxicity in primary cultures of rat cerebellar granule cells. The mGluR-evoked responses are potentiated by aniracetam, which per se also elicits neuroprotection. Aniracetam concentration-dependently counteracted glutsmate-, kainate-, or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced cell death and greatly facilitated neuroprotective response achieved by different concentrations of both quisqualate and trans-1-aminocyclopentane-1, 3-dicarboxylate. In addition, aniracetam potentiated the mGluR-coupled stimulation of phospholipase C, as revealed by the measurement of ³H-inositol phosphate formation. Thus, mGluRs could be a suitable target for novel pharmacological strategies pointing to the treatment of neurodegenerative diseases.     

Ability of recombinant interferon γ in vitro to restore the defective polymorphonuclear-cell-but not lymphocyte-mediated cytotoxic activities in patients with myelodysplastic syndromes

Summary In this study we analysed the in vitro effect of recombinant interferon on cytotoxic activities mediated by both lymphoid and polymorphonuclear cells from 16 patients with myelodysplastic syndromes. Our results indicate the inability of interferon to restore the defective natural killer activity, natural killer cells and lectin-induced cytotoxicity. On the contrary we detected a boosting effect on the depressed polymorphonuclear cell cytotoxic activities. In our view, the ability of interferon to potentiate polymorphonuclear cell lytic efficiency could support an alternative defensive pathway against either neoplastic or infectious agents.     

Changes in the ascorbate metabolism of apoplastic and symplastic spaces are associated with cell differentiation

Ascorbate levels and redox state, as well as the activities of the ascorbate related enzymes, have been analysed both in the apoplastic and symplastic spaces of etiolated pea (Pisum sativum L.) shoots during cellular differentiation. The ascorbate pool and the ascorbate oxidizing enzymes, namely ascorbate oxidase and ascorbate peroxidase, were present in both pea apoplast and symplast, whereas ascorbate free radical reductase and dehydroascorbate reductase were only present in the symplastic fractions. During cell differentiation the ascorbate redox enzymes changed in different ways, since a decrease in ascorbate levels, ascorbate peroxidase and ascorbate free radical reductase occurred from meristematic to differentiated cells, whereas ascorbate oxidase and dehydroascorbate reductase increased. The activity of secretory peroxidases has also been followed in the apoplast of meristematic and differentiating cells. These peroxidases increased their activity during differentiation. This behaviour was accompanied by changes in their isoenzymatic profiles. The analysis of the kinetic characteristics of the different peroxidases present in the apoplast suggests that the presence of ascorbate and ascorbate peroxidase in the cell wall could play a critical role in regulating the wall stiffening process during cell differentiation by interfering with the activity of secretory peroxidases.     

Dual role of TLR2 and myeloid differentiation factor 88 in a mouse model of invasive group B streptococcal disease

Toll-like receptors (TLRs) are involved in pathogen recognition by the innate immune system. Different TLRs and the adaptor molecule myeloid differentiation factor 88 (MyD88) were previously shown to mediate in vitro cell activation induced by group B streptococcus (GBS). The present study examined the potential in vivo roles of TLR2 and MyD88 during infection with GBS. When pups were infected locally with a low bacterial dose, none of the TLR2- or MyD88-deficient mice, but all of the wild-type ones, were able to prevent systemic spread of GBS from the initial focus. Bacterial burden was higher in MyD88- than in TLR2-deficient mice, indicating a more profound defect of host defense in the former animals. In contrast, a high bacterial dose induced high level bacteremia in both mutant and wild-type mice. Under these conditions, however, TLR2 or MyD88 deficiency significantly protected mice from lethality, concomitantly with decreased circulating levels of TNF-alpha and IL-6. Administration of anti-TNF-alpha Abs to wild-type mice could mimic the effects of TLR2 or MyD88 deficiency and was detrimental in the low dose model, but protective in the high dose model. In conclusion, these data highlight a dual role of TLR2 and MyD88 in the host defense against GBS sepsis and strongly suggest TNF-alpha as the molecular mediator of bacterial clearance and septic shock.     

Oxidative status in chronic hepatitis C: the influence of antiviral therapy and prognostic value of serum hydroperoxide assay

The effect of alpha-interferon (alpha-IFN) and ribavirin (RBV) treatment on oxidative status in chronic hepatitis C (CHC) is unknown. AIM: To study the time course of oxidative status in patients with CHC during alpha-IFN and RBV administration, and to evaluate the role of oxidative status in order to predict the therapeutic response. PATIENTS AND METHODS: Fifty one patients with CHC were studied. All received a combination of alpha-IFN and RBV for 6 or 12 months in relation to the type of response. The hydroperoxides concentration in serum test samples by D-ROM test was measured in all of the patients before therapy. In 27 patients, hydroperoxides were also measured during the treatment and during the 12 subsequent months. RESULTS: Cross-sectional analysis demonstrates that patients with a successive long-term response had a lower basal serum hydroperoxide concentration than non-responders (280 +/- 40.8 vs 337 +/- 83 CARR Units, p < 0.05). This resulted to be an independent factor predictive of long-term response in the multi-varied analysis. Longitudinal observation on 27 patients showed that the mean hydroperoxide concentration decreased significantly during treatment (T0 329 +/- 79.2 vs T12 272 +/- 34.5 CARR Units) and that the decrease in the mean values was mainly due to variations in the relapsers group. CONCLUSIONS: Normal basal hydroperoxide concentration helps to predict long-term response to combination therapy. The D-ROM test may be used for screening patients before treatment.