Potent Inducers of Endogenous Antimicrobial Peptides for Host Directed Therapy of Infections.

A new concept for treatment of infections is induction of our own antimicrobial peptides and the presented novel class of inducer, aroylated phenylenediamines (APDs), gives up to 20 to 30-fold induction of the human antimicrobial peptide LL-37, in vitro. In addition, oral administration of an APD in a rabbit model of Shigellosis resulted in recovery from the infection in a few days implying that APD's are promising candidates for treatment of infections.

Fast and efficient synthesis of Zorro-LNA type 3'-5'-5'-3' oligonucleotide conjugates via parallel in situ stepwise conjugation.

Zorro-LNA is a new class of therapeutic anti-gene oligonucleotides (ONs) capable of invading supercoiled DNA. The synthesis of single stranded Zorro-LNA is typically complex and laborious, requiring reverse phosphoramidites and a chemical linker connecting the two separate ON arms. Here, a simplified synthesis strategy based on 'click chemistry' is presented with a high potential for screening Zorro-LNA ONs directed against new anti-gene targets. Four different Zorro type 3'-5' 5'-3' constructs were synthesized via parallel in situ Cu(i) [3 + 2] catalysed cycloaddition. They were prepared from commercially obtained ONs functionalized on solid support (one ON with the azide and the other ON with the activated triple bond linker N-propynoylamino)-p-toluic acid (PATA)) and after cleavage from resin, they were conjugated in solution. Our report shows the benefit of combining different approaches when developing anti-gene ONs, (1) the ability for rapid and robust screening of potential targets and (2) refining the hits with more anti-gene optimized constructs. We present as well the first report showing double-strand invasion (DSI) efficiency of two combined Zorro-LNAs.

Influence of conjugation and other structural changes on the activity of Cu²âº based PNAzymes.

We have previously shown that PNA-neocuproine conjugates can act as artificial RNA restriction enzymes. In the present study we have additionally conjugated the PNA with different entities, such as oligoethers, peptides etc. and also constructed systems where the PNA is designed to clamp the target RNA forming a triplex. Some conjugations are detrimental for the activity while most are silent which means that conjugation can be done to alter physical properties without losing activity. Conjugation with a single oligoether close to the neocuproine does enhance the rate almost twofold compared to the system without the oligoether. The systems designed to clamp the RNA target by forming a triplex retain the activity if the added oligoT sequence is 5 PNA units or shorter and extends the arsenal of artificial RNA restriction enzymes. Changing the direction of a closing base pair, where the target RNA forms a bulge, from a GC to a CG pair enhances the rate of cleavage somewhat without compromising the selectivity, leading to the so far most efficient artificial nuclease reported.

Polyamine levels in breast milk are associated with mothers' dietary intake and are higher in preterm than full-term human milk and formulas.

BACKGROUND: Polyamine intake from milk is considered essential for post-natal maturation of the immune system and small intestine. The present study aimed to determine polyamine content in human milk after preterm delivery and the association with mothers' dietary intake. In comparison, the polyamine levels were compared with those in term breast milk and some corresponding formulas. METHODS: Transitional breast milk was collected from 40 mothers delivering after 24-36 weeks of gestation, and from 12 mothers delivering after full term. Food intake was assessed in mothers delivering preterm babies using a 3-day diary. Polyamines were analysed by high-performance liquid chromatography. RESULTS: The dietary intake of polyamines was significantly associated with breast milk content but weaker for spermine than for spermidine and putrescine. Total polyamine level was higher in preterm than term milk and lower in the corresponding formulas. Putrescine, spermidine and spermine contents [mean (SEM)] in preterm milk were 165.6 (25), 615.5 (80) and 167.7 (16) nmol dL⁻¹, respectively, with the levels of putrescine and spermidine being 50% and 25% higher than in term milk. The content of spermine did not differ. CONCLUSIONS: Dietary intake of polyamines has an impact on the content in breast milk. The difference between human milk after preterm and term delivery might be considered when using donor human milk for preterm infants. The corresponding formulas had lower contents. Further studies are important for determining the relationship between tissue growth and maturation and optimal intake.

Fatty acid-spermine conjugates as DNA carriers for nonviral in vivo gene delivery.

The lack of efficient in vivo gene delivery is a well-known shortcoming of nonviral delivery vectors, in particular of chemical vectors. We developed a series of novel nonviral carriers for plasmid-based in vivo gene delivery. This new transport device is based on the assembly of DNA plasmids with synthetic derivatives of naturally occurring molecules-fatty acid-spermine conjugates (or lipospermines). We tested the ability of these fatty acid conjugates to interact with plasmid DNA (pDNA) and found that they formed DNA nanocomplexes, which are protected from DNase I degradation. This protection was shown to directly correlate with the length of the aliphatic component. However, this increase in the length of the hydrocarbon chain resulted in increased toxicity. The cationic lipids used for transfection typically have a C(16) and C(18) hydrocarbon chain. Interestingly, toxicity studies, together with further characterization studies, suggested that the two most suitable candidates for in vivo delivery are those with the shortest hydrocarbon chain, butanoyl- and decanoylspermine. Morphological characterization of DNA nanocomplexes resulting from these lipospermines showed the formation of a homogenous population, with the diameter ranging approximately from 40 to 200 nm. Butanoylspermine was found to be the most promising carrier from this series, resulting in a significantly increased gene expression, in relation to naked plasmid, in both tissues herein targeted (dermis and M. tibialis anterior). Thus, we established a correlation between the in vitro properties of the ensuing DNA nanocarriers and their efficient in vivo gene expression.

Alpha-helix targeting reduces amyloid-beta peptide toxicity.

The amyloid-beta peptide (Abeta) can generate cytotoxic oligomers, and their accumulation is thought to underlie the neuropathologic changes found in Alzheimer's disease. Known inhibitors of Abeta polymerization bind to undefined structures and can work as nonspecific aggregators, and inhibitors that target conformations that also occur in larger Abeta assemblies may even increase oligomer-derived toxicity. Here we report on an alternative approach whereby ligands are designed to bind and stabilize the 13-26 region of Abeta in an alpha-helical conformation, inspired by the postulated Abeta native structure. This is achieved with 2 different classes of compounds that also reduce Abeta toxicity to cells in culture and to hippocampal slice preparations, and that do not show any nonspecific aggregatory properties. In addition, when these inhibitors are administered to Drosophila melanogaster expressing human Abeta(1-42) in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed. We conclude that stabilization of the central Abeta alpha-helix counteracts polymerization into toxic assemblies and provides a strategy for development of specific inhibitors of Abeta polymerization.

Making oligonucleotide conjugates and breaking oligonucleotides.

Presented is our most recent work on synthesis and properties of oligonucleotide analogues and conjugates These include 2'-carbamoylmethyl derivatives, peptideoligonucleotide conjugates and conjugates made to combine Watson-Crick and non-Watson-Crick interactions as well as conjugates with RNA-cleaving groups. Recent results on cleavage of RNA by artificial nucleases, is also presented.

Stabilization of RNA bulges by oligonucleotides containing 2'-naphthylmethyl-2'-deoxytubercidine.

A novel nucleoside analogue, 2'-naphthylmethyl-2'-deoxytubercidine, is synthesized and incorporated in oligonucleotides that stabilize bulges in partially complementary RNA.

Studies on the stability of dinucleoside H-phosphonates.

The stability of two dinucleoside H-phosphonates under various conditions is reported.

Evaluation of several economical computational methods for geometry optimization of phosphorus acid derivatives.

Several economical methods for geometry optimisation, applicable to larger molecules, have been evaluated for phosphorus acid derivatives. MP2/cc-pVDZ and B3LYP/6-31+G(d) geometry optimizations are used as reference points, results from geometry optimizations for other methods and their subsequent single point energy calculations are compared to these references. The geometries from HF/MIDI! optimizations were close to those of the references and subsequent single point energies with B3LYP/6-31+G(d,p) or EDF1/6-31+G(d) gave a mean average deviation (MAD) of less than 0.5 kcal mol-1 from those obtained with the reference geometries.

A method for synthesis of an artificial ribonuclease.

5-Amino-2,9-dimethyl-1,10-phenanthroline-oligonucleotide conjugates have been synthesized. A 2'-O-methyl octaribonucleotide carrying a 2'-aminoethoxymethyl linker in a central position was produced. Reaction of the aminoneocuproine phenyl carbamate with the fully deprotected oligonucleotide in aqueous solution gave virtually quantitative conversion into the conjugate. Preliminary cleavage studies in presence of zinc ions show nuclease activity towards RNA targets.

Preparation of 3'-C-branched uridine analogues, suitable for conversion into functionalised 3'-C-methylene derivatives.

A novel method for preparation of 1-[2-O-(tert-butyldimethylsilyl)-3- deoxy-3-C-hydroxymethyl-5-O-monomethoxytrityl-beta-D-ribo- pentofuranosyl]uracil by hydroboration of corresponding 3'-deoxy-3'-C-methyleneuridine derivative has been developed. Further conversion of the hydroxyl function into different leaving groups was carried out to afford derivatives suitable for conversion into various 3'-C-branched uridine analogues through substitution.

Specific metal-ion binding sites in a model of the P4-P6 triple-helical domain of a group I intron.

Divalent metal ions play a crucial role in RNA structure and catalysis. Phosphorothioate substitution and manganese rescue experiments can reveal phosphate oxygens interacting specifically with magnesium ions essential for structure and/or activity. In this study, phosphorothioate interference experiments in combination with structural sensitive circular dichroism spectroscopy have been used to probe molecular interactions underlying an important RNA structural motif. We have studied a synthetic model of the P4-P6 triple-helical domain in the bacteriophage T4 nrdB group I intron, which has a core sequence analogous to the Tetrahymena ribozyme. Rp and Sp sulfur substitutions were introduced into two adjacent nucleotides positioned at the 3' end of helix P6 (U452) and in the joining region J6/7 (U453). The effects of sulfur substitution on triple helix formation in the presence of different ratios of magnesium and manganese were studied by the use of difference circular dichroism spectroscopy. The results show that the pro-Sp oxygen of U452 acts as a ligand for a structurally important magnesium ion, whereas no such effect is seen for the pro-Rp oxygen of U452. The importance of the pro-Rp and pro-Sp oxygens of U453 is less clear, because addition of manganese could not significantly restore the triple-helical interactions within the isolated substituted model systems. The interpretation is that U453 is so sensitive to structural disturbance that any change at this position hinders the proper formation of the triple helix.

Deoxyribo- and ribonucleoside H-phosphonates.

Most methods for preparing H-phosphonate monoesters suffer from variable yields and are often incompatible with common protecting groups used in oligonucleotide synthesis. This unit describes four procedures that consistently give high yields of the desired products. Taken together, they provide an arsenal of phosphonylation procedures that it compatible with most common protecting groups.

Treasure of the Past VII: Measurement of the Thickness and Refractive Index of Very Thin Films and the Optical Properties of Surfaces by Ellipsometry.

The use of the ellipsometer for the measurement of the thickness and refractive index of very thin films is reviewed. The Poincaré sphere representation of the state of polarization of light is developed and used to describe the reflection process. Details of the operation of the ellipsometer are examined critically. A computational method is presented by which the thickness of a film of known refractive index on a reflecting substrate of known optical constants may be calculated directly from the ellipsometer readings. A method for computing both the refractive index and thickness of an unknown film is also developed. These methods have been applied to the determination of the thickness of an adsorbed water layer on chromium ferrotype plates and on gold surfaces. In the former case the thickness was 23 to 27 Å, and in the latter was 2 to 5 Å. The measurement of the thickness and refractive index of barium fluoride films evaporated on chromium ferrotype surfaces is used as an illustration of the simultaneous determination of these two quantities.

Mechanism of RNase T1: concerted triester-like phosphoryl transfer via a catalytic three-centered hydrogen bond.

BACKGROUND: The microscopic events of ribonuclease (RNase) catalyzed phosphoryl transfer reactions are still a matter of debate in which the contenders adhere to either the classical concerted acid-base mechanism or a more sequential triester-like mechanism. In the case of RNase A, small thio-effects of the nonbridging oxygens have been invoked in favor of the classical mechanism. However, the RNase T1 catalyzed transphosphorylation of phosphorothioate RNA is highly stereoselective. R(P) thio-substituted RNA is depolymerized 60000 times faster than S(P) thio-substituted RNA by this enzyme, whereas the uncatalyzed cleavage of both substrates occurs at comparable rates. We combined site-directed mutagenesis in the RNase active site and stereospecific thio-substitution of an RNA substrate to probe the intermolecular interactions of the enzyme with the nonbridging pro-S(P) oxygen that bring about this stereoselectivity of RNase T1. RESULTS: Thio-substitution of the nonbridging pro-S(P) oxygen in the substrate afflicts chemical turnover but not ground state binding whereas thio-substitution of the nonbridging pro-R(P) oxygen does not affect the kinetics of RNase T1. Site-directed mutagenesis of the catalytic base Glu58 impairs the enzyme's ability to discriminate both phosphorothioate diastereomers. Glu58Ala RNase T1 cleaves R(P) and S(P) phosphorothioate RNA with similar rates. The dependence of the pro-S(P) thio-effect on the presence of the Glu58 carboxylate evidences a strong rate-limiting interaction between the nonbridging pro-S(P) oxygen and the catalytic base Glu58 in the wild type enzyme. CONCLUSIONS: Based on these results, we put forward a new triester-like mechanism for the RNase T1 catalyzed reaction that involves a three-centered hydrogen bond between the 2'-OH group, the nonbridging pro-S(P) oxygen and one of the carboxylate oxygens of Glu58. This interaction allows nucleophilic attack on an activated phosphate to occur simultaneously with general base catalysis, ensuring concerted phosphoryl transfer via a triester-like mechanism.

Optical spectroscopic study of the effects of a single deoxyribose substitution in a ribose backbone: implications in RNA-RNA interaction.

The 2'-OH group in the ribose sugars of an RNA molecule plays an important role in guiding tertiary interactions that stabilize different RNA structural motifs. Deoxyribose, or 2'-OH by 2'-H, substitution in both the single-stranded and the duplex part of an RNA backbone has been routinely used to evaluate what role the 2'-OH plays in different tertiary interactions that guide an RNA-RNA contact. A deoxyribose substitution not only has the effect of removing a hydrogen bond donating group, but also introduces a sugar moiety with a preference for C2'-endo pucker in a backbone of predominantly C3'-endo sugars. This study evaluates the effects of a single deoxyribose substitution in both single-stranded and double-helical forms of RNA oligomers. A single-stranded, nonrepetitive 7-mer oligoribonucleotide (7-mer RNA) and four different variants having the same base sequence but with a single deoxyribose sugar at different positions in the strands have been studied by ultraviolet (UV) absorption, circular dichroism (CD), and Fourier transform infrared (FTIR) spectroscopy. Duplexes were formed by association with the complementary strand of the 7-mer RNA. The results show that both RNA and DNA single strands have preorganized conformations with spectral properties resembling those of A- and B-form helices, respectively, with RNA being more heterogeneous than its DNA counterpart. A single deoxyribose substitution perturbs the structure of the RNA backbone, with the effect being more pronounced in the single-stranded than in the duplex structure. The perturbation depends on the position of the 2'-H substitution in the strand.

A method for S- and O-palmitoylation of peptides: synthesis of pulmonary surfactant protein-C models.

A method for O- and S-palmitoylation of non-protected peptides has been developed. The peptides are treated with excess of palmitoyl chloride in 100% trifluoroacetic acid for 10 min at room temperature. The acidic conditions prevent acylation of amino groups, which is only significant after prolonged treatment (hours to days). The tripeptides Gly-Cys-Phe and Gly-Ser-Phe were converted into the respective S- and O-palmitoylated compounds, and the hydrophobic pulmonary surfactant protein-C model peptides, LRIPCCPVNLKRLLVVV [SP-C(1-17)] and FGIPSSPVLKRLLILLLLLLLILLLILGALLMGL [SP-C(Leu)] were converted into their respective S,S- and O,O-dipalmitoylated peptides. The reactions were virtually quantitative, and the palmitoylated peptides were isolated in about 75-80% yield after reversed-phase HPLC purification. CD spectroscopy showed that S, S-dipalmitoylation of SP-C(1-17) affects the peptide secondary structure (substantial increase in the alpha-helix content) in dodecylphosphocholine micelles.

Mercury inductions in persons with subjective symptoms alleged to dental amalgam fillings.

This study was carried out to determine whether health disturbances alleged to mercury release from dental amalgam fillings, i.e. "amalgam disease", may be caused by an increased sensitivity to mercury (Hg). In the form of a double-blind test, 39 volunteers who themselves suspected "amalgam disease" inhaled small doses of mercury vapour (0.6-10 microg) or pure air in a random sequence. After the induction procedure, the test persons assessed whether they reacted or not, i.e. experienced increased illness or not. The test persons also registered the daily intensity of their various symptoms. Calculated on the whole population, there was no statistically significant difference between the number of reactions after inhalation of mercury vapour compared with after inhalation of air. Two subjects, however, reacted significantly more often to mercury vapour than to air. The results do not support that short-term exposure to low doses of Hg vapour in general promotes clinical illness in subjects who themselves suspect "amalgam disease". The deviating reactions presented by two test persons, however, may support the theory that occasional individuals can be sensitive to very low doses of Hg.

Distribution of HIV type 1 (HIV-1) in blood components: detection and significance of high levels of HIV-1 associated with platelets.

BACKGROUND: Although inactivation of enveloped viruses transmitted by plasma derivatives has been successful, no methods for virus inactivation or removal have been established for platelet concentrates or red cell (RBC) components. Relatively little is known regarding the extent or significance of virus interactions with the cellular constituents in these components. STUDY DESIGN AND METHODS: Units of whole blood were collected from six HIV type 1 (HIV-1)-positive, asymptomatic individuals and separated into peripheral blood mononuclear cells (PBMNCs), cell-free plasma, white cell-reduced platelet concentrate, and white cell-reduced RBCs. DNA and RNA polymerase chain reaction and virus culture methods were used to study the compartmentalization of HIV-1 immediately after component preparation and after storage. RESULTS: As expected, HIV DNA and infectious virus were detected in fresh blood and in PBMNCs, and virion-associated RNA was detected in fresh plasma from all six donors. The levels of viral nucleic acids in these preparations remained relatively stable with 4 degrees C storage, whereas infectivity of PBMNCs was rapidly lost. Washed RBCs tested negative for HIV in all assays at all time points. Platelets retained high levels of HIV RNA (but not infectivity) after extensive washing, as well as after storage at 4 and 22 degrees C. High-level platelet-associated HIV-1 was also demonstrated in samples collected during early seroconversion. Periseroconversion and postseroconversion levels of platelet-associated HIV-1 correlated with the level of plasma viremia and with the rate of progression to AIDS. Cell-free virus from donor plasma and tissue culture fluid rapidly and firmly attached to platelets from noninfected donors. Infectivity of tissue culture virus bound to platelets was demonstrated in vitro. CONCLUSION: Significant levels of HIV-1 are associated with platelets during all stages of infection. Platelet-associated HIV could either mediate virus clearance or facilitate virus dissemination and expanded tropism. Finally, virus inactivation research must address virus associations with platelets.