Roger Strömberg

Nucleoside H-phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach

Abstract A rapid synthesis of oligodeoxyribonucleotides on solid support is described via coupling of the deoxyribonucleoside 3′- H-phosphonates in the presence of various condensing reagents.

Nucleoside H-phosphonates. IV. Automated solid phase synthesis of oligoribonucleotides by the hydrogenphosphonate approach

Abstract A rapid and efficient synthesis of oligoribonucleotides on solid support is described via coupling of 5′- O -dimethoxytrityl-2′- O -t-butyldimethylsilyl ribonucleoside 3′-H-phosphonates II to the polymer bound nucleoside in the presence of pivaloyl chloride as coupling agent.

Biological Activity and Biotechnological Aspects of Peptide Nucleic Acid

During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

Phenylbutyrate Counteracts Shigella Mediated Downregulation of Cathelicidin in Rabbit Lung and Intestinal Epithelia: A Potential Therapeutic Strategy

Background Cathelicidins and defensins are endogenous antimicrobial peptides (AMPs) that are downregulated in the mucosal epithelia of the large intestine in shigellosis. Oral treatment of Shigella infected rabbits with sodium butyrate (NaB) reduces clinical severity and counteracts the downregulation of cathelicidin (CAP-18) in the large intestinal epithelia. Aims To develop novel regimen for treating infectious diseases by inducing innate immunity, we selected sodium 4-phenylbutyrate (PB), a registered drug for a metabolic disorder as a potential therapeutic candidate in a rabbit model of shigellosis. Since acute respiratory infections often cause secondary complications during shigellosis, the systemic effect of PB and NaB on CAP-18 expression in respiratory epithelia was also evaluated. Methods The readouts were clinical outcomes, CAP-18 expression in mucosa of colon, rectum, lung and trachea (immunohistochemistry and real-time PCR) and release of the CAP-18 peptide/protein in stool (Western blot). Principal findings Significant downregulation of CAP-18 expression in the epithelia of rectum and colon, the site of Shigella infection was confirmed. Interestingly, reduced expression of CAP-18 was also noticed in the epithelia of lung and trachea, indicating a systemic effect of the infection. This suggests a causative link to acute respiratory infections during shigellosis. Oral treatment with PB resulted in reduced clinical illness and upregulation of CAP-18 in the epithelium of rectum. Both PB and NaB counteracted the downregulation of CAP-18 in lung epithelium. The drug effect is suggested to be systemic as intravenous administration of NaB could also upregulate CAP-18 in the epithelia of lung, rectum and colon. Conclusion Our results suggest that PB has treatment potential in human shigellosis. Enhancement of CAP-18 in the mucosal epithelia of the respiratory tract by PB or NaB is a novel discovery. This could mediate protection from secondary respiratory infections that frequently are the lethal causes in dysentery.

Polyamines in foods: development of a food database

Background Knowing the levels of polyamines (putrescine, spermidine, and spermine) in different foods is of interest due to the association of these bioactive nutrients to health and diseases. There is a lack of relevant information on their contents in foods. Objective To develop a food polyamine database from published data by which polyamine intake and food contribution to this intake can be estimated, and to determine the levels of polyamines in Swedish dairy products. Design Extensive literature search and laboratory analysis of selected Swedish dairy products. Polyamine contents in foods were collected using an extensive literature search of databases. Polyamines in different types of Swedish dairy products (milk with different fat percentages, yogurt, cheeses, and sour milk) were determined using high performance liquid chromatography (HPLC) equipped with a UV detector. Results Fruits and cheese were the highest sources of putrescine, while vegetables and meat products were found to be rich in spermidine and spermine, respectively. The content of polyamines in cheese varied considerably between studies. In analyzed Swedish dairy products, matured cheese had the highest total polyamine contents with values of 52.3, 1.2, and 2.6 mg/kg for putrescine, spermidine, and spermine, respectively. Low fat milk had higher putrescine and spermidine, 1.2 and 1.0 mg/kg, respectively, than the other types of milk. Conclusions The database aids other researchers in their quest for information regarding polyamine intake from foods. Connecting the polyamine contents in food with the Swedish Food Database allows for estimation of polyamine contents per portion.

Nucleoside H-Phosphonates. V. The Mechanism of Hydrogenphosphonate Diester Formation Using Acyl Chlorides as Coupling Agents in Oligonucleotide Synthesis by the Hydrogenphosphonate Approach

The H-phosphono-acyl mixed anhydrides of type III were found to be the main intermediates during H-phosphonate diester formation using acyl chlorides as coupling agents in the reaction of hydrogenp...

An approach towards the synthesis of oligomers containing a N-2-hydroxyethyl-aminomethylphosphonate backbone: A novel PNA analogue

A convenient route to the preparation of 4-methoxy-1-oxido-pyridine-2-methyl N-2-(4,4′-dimethoxytrityloxy)ethyl-N-thymin-1-yl-aminomethylphosphonate (1a, T∗) and the corresponding N4-benzoylcytosin-1-yl derivative 1b (C∗) is reported. These PPNA monomers proved to be suitable building blocks in a solid-support synthesis of the tetradecameric fragment (C∗T∗T∗T∗C∗T∗T∗T∗T∗C∗T∗C∗T∗)dT.

PNAzymes That Are Artificial RNA Restriction Enzymes

DNA-cleaving restriction enzymes are well-known tools in biomedical and biotechnological research. There are, however, no corresponding enzymes known for RNA cleavage. There has been an ongoing development of artificial ribonucleases, including some attempts at sequence selectivity. However, so far these systems have displayed modest rates of cleavage, and in most cases, the cleaver has been used in excess or in stoichiometric amounts. In the current work, we present PNA-based systems (PNAzymes) that carry a Cu(II)-2,9-dimethylphenanthroline group and that act as site and sequence specific RNases. The general basis for the systems is that the target is cleaved at a nonbase paired region (RNA bulge) which is formed in the substrate upon binding of the PNAzyme. With this copper based system, cleavage takes place at virtually only one site and with a half-life of down to 30 min under stoichiometric conditions. Efficient turnover of RNA-substrate is shown with a 100-fold excess of substrate, thus, demonstrating true enzyme behavior. In addition, alteration of the sequence in the RNA bulge or a mismatch in the base-pairing region leads to substantial decreases in rate showing both kinetic resolution and binding discrimination in the substrate selectivity. The selectivity is further demonstrated by the substrates, with two potential cleavage sites differing in only one base, are cleaved only at the site that either does not have a mismatch or is kinetically preferred. We suggest that these systems can serve as a basis for construction of RNA restriction enzymes for in vitro manipulations.

Alpha-helix targeting reduces amyloid-beta peptide toxicity.

The amyloid-β peptide (Aβ) can generate cytotoxic oligomers, and their accumulation is thought to underlie the neuropathologic changes found in Alzheimers disease. Known inhibitors of Aβ polymerization bind to undefined structures and can work as nonspecific aggregators, and inhibitors that target conformations that also occur in larger Aβ 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 Aβ in an α-helical conformation, inspired by the postulated Aβ native structure. This is achieved with 2 different classes of compounds that also reduce Aβ 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 Aβ1–42 in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed. We conclude that stabilization of the central Aβ α-helix counteracts polymerization into toxic assemblies and provides a strategy for development of specific inhibitors of Aβ polymerization.

Lactose in human breast milk an inducer of innate immunity with implications for a role in intestinal homeostasis.

Postpartum, infants have not yet established a fully functional adaptive immune system and are at risk of acquiring infections. Hence, newborns are dependent on the innate immune system with its antimicrobial peptides (AMPs) and proteins expressed at epithelial surfaces. Several factors in breast milk are known to confer immune protection, but which the decisive factors are and through which manner they work is unknown. Here, we isolated an AMP-inducing factor from human milk and identified it by electrospray mass spectrometry and NMR to be lactose. It induces the gene (CAMP) that encodes the only human cathelicidin LL-37 in colonic epithelial cells in a dose- and time-dependent manner. The induction was suppressed by two different p38 antagonists, indicating an effect via the p38-dependent pathway. Lactose also induced CAMP in the colonic epithelial cell line T84 and in THP-1 monocytes and macrophages. It further exhibited a synergistic effect with butyrate and phenylbutyrate on CAMP induction. Together, these results suggest an additional function of lactose in innate immunity by upregulating gastrointestinal AMPs that may lead to protection of the neonatal gut against pathogens and regulation of the microbiota of the infant.