Mechanism of hcnA mRNA recognition in the Gac/Rsm signal transduction pathway of Pseudomonas fluorescens.

In the plant-beneficial bacterium Pseudomonas fluorescens CHA0, the expression of antifungal exoproducts is controlled by the GacS/GacA two-component system. Two RNA binding proteins (RsmA, RsmE) ensure effective translational repression of exoproduct mRNAs. At high cell population densities, GacA induces three small RNAs (RsmX, RsmY, RsmZ) which sequester both RsmA and RsmE, thereby relieving translational repression. Here we systematically analyse the features that allow the RNA binding proteins to interact strongly with the 5' untranslated leader mRNA of the P. fluorescens hcnA gene (encoding hydrogen cyanide synthase subunit A). We obtained evidence for three major RsmA/RsmE recognition elements in the hcnA leader, based on directed mutagenesis, RsmE footprints and toeprints, and in vivo expression data. Two recognition elements were found in two stem-loop structures whose existence in the 5' leader region was confirmed by lead(II) cleavage analysis. The third recognition element, which overlapped the hcnA Shine-Dalgarno sequence, was postulated to adopt either an open conformation, which would favour ribosome binding, or a stem-loop structure, which may form upon interaction with RsmA/RsmE and would inhibit access of ribosomes. Effective control of hcnA expression by the Gac/Rsm system appears to result from the combination of the three appropriately spaced recognition elements.

Solid-Phase Synthesis of New Trp(Nps)-Containing Dipeptide Derivatives as TRPV1 Channel Blockers

Trp(Nps)-Lys-NH2 derivatives, bearing alkyl or guanidine groups either at the N-terminus or on the Lys side-chain or at both positions were conveniently prepared on solid-phase and evaluated as TRPV1 channel antagonists.

Nucleophile-Catalyzed Additions to Activated Triple Bonds. Protection of Lactams, Imides, and Nucleosides with MocVinyl and Related Groups

Additions of lactams, imides, (S)-4-benzyl-1,3-oxazolidin-2-one, 2-pyridone, pyrimidine-2,4-diones (AZT derivatives), or inosines to the electron-deficient triple bonds of methyl propynoate, tert-butyl propynoate, 3-butyn-2-one, N-propynoylmorpholine, or N-methoxy-N-methylpropynamide in the presence of many potential catalysts were examined. DABCO and, second, DMAP appeared to be the best (highest reaction rates and E/Z ratios), while RuCl3, RuClCp*(PPh3)2, AuCl, AuCl(PPh3), CuI, and Cu2(OTf)2 were incapable of catalyzing such additions. The groups incorporated (for example, the 2-(methoxycarbonyl)ethenyl group that we name MocVinyl) serve as protecting groups for the above-mentioned heterocyclic CONH or CONHCO moieties. Deprotections were accomplished via exchange with good nucleophiles: the 1-dodecanethiolate anion turned out to be the most general and efficient reagent, but in some particular cases other nucleophiles also worked (e.g., MocVinyl-inosines can be cleaved with succinimide anion). Some structural and mechanistic details have been accounted for with the help of DFT and MP2 calculations.

Constrained peptidomimetics reveal detailed geometric requirements of covalent prolyl oligopeptidase inhibitors.

Prolyl oligopeptidases cleave peptides on the carboxy side of internal proline residues and their inhibition has potential in the treatment of human brain disorders. Using our docking program fitted, we have designed a series of constrained covalent inhibitors, built from a series of bicyclic scaffolds, to study the optimal shape required for these small molecules. These structures bear nitrile functional groups that we predicted to covalently bind to the catalytic serine of the enzyme. Synthesis and biological assays using human brain-derived astrocytic cells and endothelial cells and human fibroblasts revealed that these compounds act as selective inhibitors of prolyl oligopeptidase activity compared to prolyl-dipeptidyl-aminopeptidase activity, are able to penetrate the cells and inhibit intracellular activities in intact living cells. This integrated computational and experimental study shed light on the binding mode of inhibitors in the enzyme active site and will guide the design of future drug-like molecules.

Role of Mitogen-Activated Protein Kinases in Myocardial Ischemia-Reperfusion Injury during Heart Transplantation.

In solid organ transplantation, ischemia/reperfusion (IR) injury during organ procurement, storage and reperfusion is an unavoidable detrimental event for the graft, as it amplifies graft inflammation and rejection. Intracellular mitogen-activated protein kinase (MAPK) signaling pathways regulate inflammation and cell survival during IR injury. The four best-characterized MAPK subfamilies are the c-Jun NH2-terminal kinase (JNK), extracellular signal- regulated kinase-1/2 (ERK1/2), p38 MAPK, and big MAPK-1 (BMK1/ERK5). Here, we review the role of MAPK activation during myocardial IR injury as it occurs during heart transplantation. Most of our current knowledge regarding MAPK activation and cardioprotection comes from studies of preconditioning and postconditioning in nontransplanted hearts. JNK and p38 MAPK activation contributes to myocardial IR injury after prolonged hypothermic storage. p38 MAPK inhibition improves cardiac function after cold storage, rewarming and reperfusion. Small-molecule p38 MAPK inhibitors have been tested clinically in patients with chronic inflammatory diseases, but not in transplanted patients, so far. Organ transplantation offers the opportunity of starting a preconditioning treatment before organ procurement or during cold storage, thus modulating early events in IR injury. Future studies will need to evaluate combined strategies including p38 MAPK and/or JNK inhibition, ERK1/2 activation, pre- or postconditioning protocols, new storage solutions, and gentle reperfusion.

Nucleophile-Catalyzed Additions to Activated Triple Bonds. Protection of Lactams, Imides, and Nucleosides with MocVinyl and Related Groups

Additions of lactams, imides, (S)-4-benzyl-1,3-oxazolidin-2-one, 2-pyridone, pyrimidine-2,4-diones (AZT derivatives), or inosines to the electron-deficient triple bonds of methyl propynoate, tert-butyl propynoate, 3-butyn-2-one, N-propynoylmorpholine, or N-methoxy-N-methylpropynamide in the presence of many potential catalysts were examined. DABCO and, second, DMAP appeared to be the best (highest reaction rates and E/Z ratios), while RuCl3, RuClCp*(PPh3)2, AuCl, AuCl(PPh3), CuI, and Cu2(OTf)2 were incapable of catalyzing such additions. The groups incorporated (for example, the 2-(methoxycarbonyl)ethenyl group that we name MocVinyl) serve as protecting groups for the above-mentioned heterocyclic CONH or CONHCO moieties. Deprotections were accomplished via exchange with good nucleophiles: the 1-dodecanethiolate anion turned out to be the most general and efficient reagent, but in some particular cases other nucleophiles also worked (e.g., MocVinyl-inosines can be cleaved with succinimide anion). Some structural and mechanistic details have been accounted for with the help of DFT and MP2 calculations.

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.

Preparation of benzolactams by Pd(II)-catalyzed carbonylation of N-unprotected arylethylamines

An unprecedented NH2-directed Pd(II)-catalytic carbonylation of quaternary aromatic α -amino esters to yield 6-membered 10 benzolactams has been developed. The reaction shows a strong bias to 6-membered lactams over 5-membered ones. The steric hindrance around the amino group seems to be pivotal for the success of the process.

JNK controls the onset of mitosis of planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

Coiodação de alquenos com nucleófilos oxigenados: reações intermoleculares

A review on the electrophilic addition of iodine to alkenes in the presence of oxygen containing nucleophiles (cohalogenation reaction) is presented. The intermolecular reactions are discussed with emphasis in methods of reaction and synthetic applications of the resulting vicinal iodo-functionalized products (iodohydrins, beta-iodoethers and beta-iodocarboxylates).

Câncer e agentes antineoplásicos ciclo-celular específicos e ciclo-celular não específicos que interagem com o DNA: uma introdução

The chemotherapy agents against cancer may be classified as "cell cycle-specific" or "cell cycle-nonspecific". Nevertheless, several of them have their biological activity related to any kind of action on DNA such as: antimetabolic agents (DNA synthesis inhibition), inherently reactive agents (DNA alkylating electrophilic traps for macromolecular nucleophiles from DNA through inter-strand cross-linking - ISC - alkylation) and intercalating agents (drug-DNA interactions inherent to the binding made due to the agent penetration in to the minor groove of the double helix). The earliest and perhaps most extensively studied and most heavily employed clinical anticancer agents in use today are the DNA inter-strand cross-linking agents.

Reatividade de ciclopropenonas frente a nucleófilos e sua correlação com potenciais de redução em meio aprótico

In this work we studied the reactivity of isopropylphenylcyclopropenone towards some nitrogen nucleophiles whose reactions with methylphenylcyclopropenone and diphenylcyclopropenone were previously studied. The electrochemical behavior of these cyclopropenones was evaluated for the first time, and a correlation between electrochemical parameters and reactivity of this class of compounds was done.

Preparação e caracterização de blendas de quitosana/poli(álcool vinílico) reticuladas quimicamente com glutaraldeído para aplicação em engenharia de tecido

In this study, novel Chitosan/PVA based films were chemically crosslinked by glutaraldehyde, under pH=(4,00 ±0,05), in order to achieve structures tailored for wound tissue engineering applications. Both precursors and developed films were characterized by FTIR, SEM and XRD in order to determine the presence of chemicals groups and nanostructural order, respectively. The results have shown that the glutaraldehyde crosslinking have altered the crystallinity of pure chitosan and the increase on the C=N bands and simultaneous decrease on NH2 bands suggested that Chitosan/GA crosslinking has preference to occur in carbon-2 of the saccharide ring by the Schiff's base reaction. Also, FTIR spectroscopy clearly showed that crosslinking has also taken place with blends of PVA and chitosan. The mechanical properties presented high degree dependence with on the increase of the content of chitosan and glutaraldehyde. The results have indicated that, by controlling the ratio [PVA]/[chitosan] in the blends and the extent of chemical crosslinking, it was possible to tailor the hybrid network produced aiming to obtain properties of interest for the specific application.

Preparação de N-alquil derivados do 1-bromo-2,4-dinitrobenzeno e do 1-cloro-2-nitrobenzeno: Uma alternativa às aulas práticas de substituição nucleofílica aromática

Preparation of the title compounds are described as an alternative nucleophilic aromatic substitution for practices in the graduate laboratory. The low toxicity and disponibility of the reagents make a suitable procedure approach to experiments regarding this aromatic reaction.

Estudo teórico de propriedades ópticas não-lineares de nanotubos de carbono de parede única quimicamente modificados

Structure and first hyperpolarizability for a series of armchair a(5,5) chemically modified carbon nanotubes (CNT) were calculated at semiempirical and density functional levels of theory. The 4,4´-substituted stilbenes were selected as chromophore with substituents at position 4´ set to X=NO2, H, Cl, OH and NH2. The calculated values for static first hyperpolarizability (β) were almost linearly dependent on the electronic effect of the group X, increasing from NO2 to NH2. At DFT level the effect of inserting the chromophore in the CNT surface was to enhance the β value up to 70% relative to the free 4,4´-substituted stilbene.