Novel lactoferrampin antimicrobial peptides derived from human lactoferrin

Human lactoferrampin is a novel antimicrobial peptide found in the cationic N-terminal lobe of the iron-binding human lactoferrin protein. The amino acid sequence that directly corresponds to the previously characterized bovine lactoferrin-derived lactoferrampin peptide is inactive on its own (WNLLRQAQEKFGKDKSP, residues 269-285). However, by increasing the net positive charge near the C-terminal end of human lactoferrampin, a significant increase in its antibacterial and Candidacidal activity was obtained. Conversely, the addition of an N-terminal helix cap (sequence DAI) did not have any appreciable effect on the antibacterial or antifungal activity of human lactoferrampin peptides, even though it markedly influenced that of bovine lactoferrampin. The solution structure of five human lactoferrampin variants was determined in SDS micelles and all of the structures display a well-defined amphipathic N-terminal helix and a flexible cationic C-terminus. Differential scanning calorimetry studies indicate that this peptide is capable of inserting into the hydrophobic core of a membrane, while fluorescence spectroscopy results suggest that a hydrophobic patch encompassing the single Trp and Phe residues as well as Leu, Ile and Ala side chains mediates the interaction between the peptide and the hydrophobic core of a phospholipid bilayer.     

S-Adenosyl-N-decyl-aminoethyl, a Potent Bisubstrate Inhibitor of Mycobacterium tuberculosis Mycolic Acid Methyltransferases

S-Adenosylmethionine-dependent methyltransferases (AdoMet-MTs) constitute a large family of enzymes specifically transferring a methyl group to a range of biologically active molecules. Mycobacterium tuberculosis produces a set of paralogous AdoMet-MTs responsible for introducing key chemical modifications at defined positions of mycolic acids, which are essential and specific components of the mycobacterial cell envelope. We investigated the inhibition of these mycolic acid methyltransferases (MA-MTs) by structural analogs of the AdoMet cofactor. We found that S-adenosyl-N-decyl-aminoethyl, a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain, inhibited MA-MTs from Mycobacterium smegmatis and M. tuberculosis strains, both in vitro and in vivo, with IC₅₀ values in the submicromolar range. By contrast, S-adenosylhomocysteine, the demethylated reaction product, and sinefungin, a general AdoMet-MT inhibitor, did not inhibit MA-MTs. The interaction between Hma (MmaA4), which is strictly required for the biosynthesis of oxygenated mycolic acids in M. tuberculosis, and the three cofactor analogs was investigated by x-ray crystallography. The high resolution crystal structures obtained illustrate the bisubstrate nature of S-adenosyl-N-decyl-aminoethyl and provide insight into its mode of action in the inhibition of MA-MTs. This study has potential implications for the design of new drugs effective against multidrug-resistant and persistent tubercle bacilli.One-third of the world population is infected with the tubercle bacillus, Mycobacterium tuberculosis, and tuberculosis kills one person every 20 s. The inhaled pathogenic bacilli are taken up by phagocytosis by pulmonary macrophages, which, together with lymphocytes and dendritic cells, form granulomas. The bacilli persist in the granuloma until their reactivation, dissemination into the lungs, and the triggering of disease. The natural resistance of persistent tubercle bacilli to drugs and the emergence of multidrug-resistant and extensively drug-resistant M. tuberculosis strains are two main concerns in the treatment of the disease. A survey carried out by the Centers for Disease Control and Prevention and the World Health Organization between 2000 and 2004 reported that 20% of 17,690 M. tuberculosis isolates from 49 countries were multidrug-resistant, and 2% were extensively drug-resistant (1). The development of new drugs effective against persistent and drug-resistant bacilli has therefore become a priority.The thick lipid-rich envelope of the Mycobacterium genus is characterized by the presence of mycolic acids (MAs),⁴ very long chain (C₆₀–C₉₀) α-alkylated β-hydroxylated fatty acids (2). MAs are the major components of the mycomembrane (3, 4) lipid bilayer, which plays a key role in both the architecture and permeability of the mycobacterial envelope. The MA biosynthetic pathway is essential for mycobacterial survival. MAs are generated by Claisen condensation between two fatty acyl chains as follows: the very long meromycoloyl chain (C₄₀–C₆₀) and a shorter saturated chain (C₂₂–C₂₆) (2). The different types of MAs are defined by the presence of decorations introduced at proximal and distal positions of the meromycolic chain (Fig. 1A) by a family of paralogous S-adenosylmethionine-dependent methyltransferases (AdoMet-MTs), the mycolic acid methyltransferases (MA-MTs). These chemical modifications are known to be important for the pathogenicity, virulence, and persistence of M. tuberculosis. For example, the cis-cyclopropane introduced at the proximal position of α-MAs by PcaA has an impact on the persistence of the tubercle bacillus within infected organisms (5). Furthermore, the keto and methoxy groups, with a vicinal methyl ramification at the distal position of oxygenated MAs, play a role in M. tuberculosis virulence in the mouse model of infection (6) and have recently been reported to be involved in host-pathogen interplay. Indeed, oxygenated MAs have been shown to modulate IL-12p40 production by macrophages (7) and to trigger the in vitro differentiation of monocyte-derived macrophages into foamy macrophages, which house the bacillus in a dormant state, within granulomas (8). Oxygenated MA biosynthesis requires the Hma (MmaA4) methyltransferase (Fig. 1B), as demonstrated by the absence of the oxygenated form in an M. tuberculosis hma knock-out mutant (6, 9). These results suggest that the enzymes responsible for adding the decorations to MAs, including oxygenated groups in particular, may be relevant pharmacological targets for the development of new antituberculous drugs (10).Open in a separate windowFIGURE 1.A, structures of MAs from M. tuberculosis and M. smegmatis. D, distal position; P, proximal position. Enzymes involved in the introduction of decorations on the meromycolic chain are indicated. B, proposed reaction scheme for the introduction of oxygenated groups. m = 17, 19; n, unknown; X, unknown carrier.Based on the essential role played by MA-MTs in the physiopathology of tuberculosis, several studies have investigated the possible inhibition of this family of enzymes. A recent study revealed that the antituberculous drug thiacetazone and its chemical analogs inhibited MA cyclopropanation at concentrations in the micromolar range (11). Another study, based on mixtures of crude extracts of heat-inactivated mycobacteria and recombinant Escherichia coli overproducing MA-MTs, suggested that the incorporation of [³H]AdoMet into growing meromycolic chains is inhibited by a high concentration (1 mg/ml, i.e. 2.6 mm) of S-adenosyl-l-homocysteine (AdoHcy) or sinefungin (12), the demethylated reaction product and a natural structural analog of AdoMet, respectively (Fig. 2). By contrast, AdoHcy and sinefungin are strong inhibitors of other AdoMet-MTs in vitro, including the cyclopropane fatty-acid synthase (CFAS) from E. coli (K_i of 30 and 0.22 μm, respectively) (13, 14). However, they are active only against the isolated enzyme, whereas S-adenosyl-N-decyl-aminoethyl (SADAE), a molecule in which the amino acid moiety of AdoMet is substituted by a lipid chain (Fig. 2), is active against CFAS both in vitro (K_i,app = 6 μm) and in vivo (complete inhibition at 150 μm) (15). The broad screening of possible inhibitors of MA-MTs with an in vitro mini-assay poses a major challenge, as these enzymes most likely use very long meromycolic chains as substrates. In this context, the similarity between CFAS and Hma in terms of their sequences (31% sequence identity) and substrates may be useful, as it suggests that SADAE may inhibit MA-MTs (15).Open in a separate windowFIGURE 2.Structure of AdoMet and of the AdoHcy, sinefungin, and SADAE analogs.We report here our investigations of the interactions between Hma and SADAE, as compared with those between Hma and AdoHcy or sinefungin, and the potential impact of these interactions on the activities of Hma and other MA-MTs and mycobacterial growth. Our high resolution crystallographic characterization of the Hma-SADAE interaction illustrates the bisubstrate nature of the ligand, which is strongly correlated with its strong inhibitory properties.     

Chemicals Possessing a Neurotrophin-Like Activity on Dopaminergic Neurons in Primary Culture

Background

Neurotrophic factors have been shown to possess strong neuroprotective and neurorestaurative properties in Parkinson''s disease patients. However the issues to control their delivery into the interest areas of the brain and their surgical administration linked to their unability to cross the blood brain barrier are many drawbacks responsible of undesirable side effects limiting their clinical use. A strategy implying the use of neurotrophic small molecules could provide an interesting alternative avoiding neurotrophin administration and side effects. In an attempt to develop drugs mimicking neurotrophic factors, we have designed and synthesized low molecular weight molecules that exhibit neuroprotective and neuritogenic potential for dopaminergic neurons.

Principal Findings

A cell-based screening of an in-house quinoline-derived compound collection led to the characterization of compounds exhibiting both activities in the nanomolar range on mesencephalic dopaminergic neurons in spontaneous or 1-methyl-4-phenylpyridinium (MPP⁺)-induced neurodegeneration. This study provides evidence that rescued neurons possess a functional dopamine transporter and underlines the involvement of the extracellular signal-regulated kinase 1/2 signaling pathway in these processes.

Conclusion

Cell-based screening led to the discovery of a potent neurotrophic compound possessing expected physico-chemical properties for blood brain barrier penetration as a serious candidate for therapeutic use in Parkinson disease.     

Lipid-Induced ER Stress: Synergistic Effects of Sterols and Saturated Fatty Acids

Stress within the endoplasmic reticulum (ER) induces a coordinated response, namely the unfolded protein response (UPR), devoted to helping the ER cope with the accumulation of misfolded proteins. Failure of the UPR plays an important role in several human diseases. Recent studies report that intracellular accumulation of saturated fatty acids (SFAs) and cholesterol, seen in diseases of high incidence, such as obesity or atherosclerosis, results in ER stress. In the present study, we evaluated the effects of perturbations to lipid homeostasis on ER stress/UPR induction in the model eukaryote Saccharomyces cerevisiae . We show that SFA originating from either endogenous (preclusion of fatty acid desaturation) or exogenous (feeding with extracellular SFA) sources trigger ER stress and that ergosterol, the major sterol in yeast, acts synergistically with SFA in this process. This latter effect is connected to ergosterol accumulation within microsomal fractions from SFA-accumulating cells, which display highly saturated phospholipid content. Moreover, treating the cells with the molecular chaperone 4-phenyl butyrate abolishes UPR induction, suggesting that lipid-induced ER stress leads to an overload of misfolded protein that acts, in turn, as the molecular signal for induction of the UPR. The present data are discussed in the context of human diseases that involve lipid deregulation.     

Heterotrophy in Tropical Scleractinian Corals

The dual character of corals, that they are both auto- and heterotrophs, was recognized early in the twentieth Century. It is generally accepted that the symbiotic association between corals and their endosymbiotic algae (called zooxanthellae) is fundamental to the development of coral reefs in oligotrophic tropical oceans because zooxanthellae transfer the major part of their photosynthates to the coral host (autotrophic nutrition). However, numerous studies have confirmed that many species of corals are also active heterotrophs, ingesting organisms ranging from bacteria to mesozooplankton. Heterotrophy accounts for between 0 and 66% of the fixed carbon incorporated into coral skeletons and can meet from 15 to 35% of daily metabolic requirements in healthy corals and up to 100% in bleached corals. Apart from this carbon input, feeding is likely to be important to most scleractinian corals, since nitrogen, phosphorus, and other nutrients that cannot be supplied from photosynthesis by the coral's symbiotic algae must come from zooplankton capture, particulate matter or dissolved compounds. A recent study showed that during bleaching events some coral species, by increasing their feeding rates, are able to maintain and restore energy reserves.
This review assesses the importance and effects of heterotrophy in tropical scleractinian corals. We first provide background information on the different food sources (from dissolved organic matter to meso- and macrozooplankton). We then consider the nutritional inputs of feeding. Finally, we review feeding effects on the different physiological parameters of corals (tissue composition, photosynthesis and skeletal growth).     

Transient Neuronal Populations Are Required to Guide Callosal Axons: A Role for Semaphorin 3C

The corpus callosum (CC) is the main pathway responsible for interhemispheric communication. CC agenesis is associated with numerous human pathologies, suggesting that a range of developmental defects can result in abnormalities in this structure. Midline glial cells are known to play a role in CC development, but we here show that two transient populations of midline neurons also make major contributions to the formation of this commissure. We report that these two neuronal populations enter the CC midline prior to the arrival of callosal pioneer axons. Using a combination of mutant analysis and in vitro assays, we demonstrate that CC neurons are necessary for normal callosal axon navigation. They exert an attractive influence on callosal axons, in part via Semaphorin 3C and its receptor Neuropilin-1. By revealing a novel and essential role for these neuronal populations in the pathfinding of a major cerebral commissure, our study brings new perspectives to pathophysiological mechanisms altering CC formation.     

A nonradioactive iodide uptake assay for sodium iodide symporter function

The standard assay for sodium iodide symporter (NIS) function is based on the measurement of radioiodide uptake (¹²⁵I) in NIS-expressing cells. However, cost and safety issues have limited the method from being used widely. Here we describe a simple spectrophotometric assay for the determination of iodide accumulation in rat thyroid-derived cells (FRTL5) based on the catalytic effect of iodide on the reduction of yellow cerium(IV) to colorless cerium(III) in the presence of arsenious acid (Sandell-Kolthoff reaction). The assay is fast and highly reproducible with a Z′ factor of 0.70. This procedure allows the screening of more than 800 samples per day and can easily be adapted to robotic systems for high-throughput screening of NIS function modulators. Using this method, the potency of several known inhibitors of NIS function was evaluated in a single day with high accuracy and reliability. Measured IC₅₀ values were essentially identical to those determined using Na¹²⁵I.     

Cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives as antioxidants against light-induced retinal degeneration

Two cholesterol-based α-phenyl-N-tert-butyl nitrone derivatives were synthesized as antioxidants against light-induced retinal degeneration. Whereas nitrone 10 significantly protected retina against bright fluorescent light exposure when injected into the vitreous at 1 mM, no protection was observed with nitrone 6. The parent compound α-phenyl-N-tert-butyl nitrone also exhibited protective activity at 9 mM but not at 1 mM. This suggests that nitrone 10 may be a candidate for the treatment of retinal diseases.