Chemical constituents from the fruiting bodies of Cryptoporus volvatus.

New drimane-type sesquiterpene cryptoporol A (1), cryptoporic acid derivative 6'-cryptoporic acid E methyl ester (2), and pseudouridine derivative cryptoporine A (3), as well as a known ergosterol 5α,8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol (4), were isolated from a 90 % alcohol extract of the fruiting bodies of Cryptoporus volvatus. The structures of these compounds were established by spectroscopic analysis and circular dichroism. 5α,8α-epidioxy-22E-ergosta-6,22-dien-3ß-ol (4) exhibited antiviral activity against porcine reproductive and respiratory syndrome virus, and all compounds showed weak antioxidant activities.

C-Nucleosides To Be Revisited.

Two new C-nucleoside analogues, BCX4430, an imino-C-nucleoside, and GS-6620, a phosphoramidate derivative of 1'-cyano-2'-C-methyl-4-aza-7,9-dideazaadenosine C-nucleoside, have been recently described as effective against filovirus infections (Marburg) and hepatitis C virus (HCV), respectively. The first C-nucleoside analogues were described about half a century ago. The C-nucleoside pseudouridine is a natural component of RNA, and various other C-nucleoside analogues have been reported previously for their antiviral and/or anticancer potential, the most prominent being pyrazofurin, tiazofurin, and selenazofurin. In the meantime, showdomycin, formycin, and various triazole, pyrazine, pyridine, dihydroxyphenyl, thienopyrimidine, pyrazolotriazine, and porphyrin C-nucleoside analogues have been described. It would be worth revisiting these C-nucleosides and derivatives thereof, including their phosphoramidates, for their therapeutic potential in the treatment of virus infections and, where appropriate, cancer as well.

Defining optimized properties of modified mRNA to enhance virus- and DNA- independent protein expression in adult stem cells and fibroblasts.

BACKGROUND: By far, most strategies for cell reprogramming and gene therapy are based on the introduction of DNA after viral delivery. To avoid the high risks accompanying these goals, non-viral and DNA-free delivery methods for various cell types are required. METHODS: Relying on an initially established PCR-based protocol for convenient template DNA production, we synthesized five differently modified EGFP mRNA (mmRNA) species, incorporating various degrees of 5-methylcytidine-5'-triphosphate (5mC) and pseudouridine-5'-triphosphate (Ψ). We then investigated their effect on i) protein expression efficiencies and ii) cell viability for human mesenchymal stem cells (hMSCs) and fibroblasts from different origins. RESULTS: Our protocol allows highly efficient mmRNA production in vitro, enabling rapid and stable protein expression after cell transfection. However, our results also demonstrate that the terminally optimal modification needs to be defined in pilot experiments for each particular cell type. Transferring our approach to the conversion of fibroblasts into skeletal myoblasts using mmRNA encoding MyoD, we confirm the huge potential of mmRNA based protein expression for virus- and DNA-free reprogramming strategies. CONCLUSION: The achieved high protein expression levels combined with good cell viability not only in fibroblasts but also in hMSCs provides a promising option for mmRNA based modification of various cell types including slowly proliferating adult stem cells. Therefore, we are confident that our findings will substantially contribute to the improvement of efficient cell reprogramming and gene therapy approaches.

Beta-pseudouridine, a beer component, reduces radiation-induced chromosome aberrations in human lymphocytes.

We previously found that drinking beer reduces chromosome aberrations in blood lymphocytes that were collect and irradiated in vitro. In this study, human whole-blood was in vitro exposed to 200kVp X-rays or 50keV/microm carbon ions in the presence or absence of beer, ethanol and beta-pseudouridine (one of the beer components). All three agents reduced the chromosome aberrations (dicentric) caused by either X-rays or carbon ions of 4Gy. The maximum protection for X-rays by ethanol, beer and beta-pseudouridine was 64, 26 and 34%, respectively, while the maximum protection for carbon-ion by ethanol, beer and beta-pseudouridine was 22, 26 and 32%, respectively. It is concluded that beta-pseudouridine, a nucleoside present in tRNA, is a potent protector against damage caused by radiation with direct and indirect actions.

Pseudouridine, an antimutagenic substance in beer towards N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).

Previously we reported that beer is antimutagenic against several food-derived mutagens including heterocyclic amines. We describe here the isolation and identification of pseudouridine from beer as an antimutagenic substance against N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). All of the 17 samples of beer tested showed inhibition of the MNNG mutagenicity in Salmonella. Extensive fractionation through chromatography of the active components from a freeze-dried beer sample gave six antimutagenic fractions. One contained pseudouridine, as characterized by the UV spectra, nuclear magnetic resonance, and co-chromatography in HPLC. Pure pseudouridine inhibited the mutagenicity of MNNG in a dose-dependent manner. The amount of pseudouridine in the beer sample, estimated at about 0.4 mg/100 ml beer, can account for 3% of the total antimutagenicity of beer. Thus, the major active components in beer remain to be identified. The role of pseudouridine in inhibiting the mutagenicity of MNNG is to be studied further. Among analogs of pseudouridine, spongouridine, but not uridine, was antimutagenic against MNNG. The bacterial mutagenicity of another methylating agent N-methyl-N-nitrosourea was also inhibited by pseudouridine. Pseudouridine is the first example among nucleosides to be shown to possess an antimutagenic property.

Cloning, expression and crystallization of pyrimidine nucleoside phosphorylase from Bacillus stearothermophilus.

Pyrimidine nucleoside phosphorylase (PYNP) from B. stearothermophilus has been cloned and purified for crystallization. Crystals of a potential protein-inhibitor complex have been prepared by co-crystallization techniques using the substrate analog pseudouridine. These crystals provide good-quality diffraction images to 2.7 A and belong to space group P21. The asymmetric unit contains the dimer structure of PYNP with unit-cell parameters a = 53.9, b = 71.9, c = 123.3 A and beta = 96.9 degrees.

The effect of pseudouridine and pH on the structure and dynamics of the anticodon stem-loop of tRNA(Lys,3).

The anticodon stem-loop hairpin of tRNA(Lys,3) was synthesized and the solution structure determined by NMR spectroscopy. The hairpin is thermodynamically stabilized by pseudouridine as determined by UV Tm measurements, and the local loop structure is stabilized with base-stacking of the nucleosides in the anticodon loop 5' of the psi 39 nucleoside modification. The tRNA(Lys,3) hairpin also contains an A(+)-C base-pair that effectively reduces the size of the normal 7 nucleotide anticodon loop to 5 nucleotides and induces a change in the loop backbone conformation. The stabilizing effects of the A(+)-C base-pair and pseudouridine are only partially additive, suggesting that the conformational changes caused by each modification are not completely compatible. The structure of the anticodon loop is distinctly different from that seen for other tRNAs exemplified by tRNA(Phe), suggesting that the full complement of modified nucleosides present in tRNA(Lys,3) should significantly change the structure compared to the unmodified tRNA anticodon loop. The conformation of the loop has important implications for the role of nucleoside modification in codon-anticodon recognition and for utilization of tRNA(Lys,3) by HIV-1 as the natural reverse transcriptase primer.

Inhibition of glucose utilization in isolated rat soleus muscle by pseudouridine: implications for renal failure.

Pseudouridine (psi) is an outstanding nucleoside which is not rebuilt into the tRNA once the parent tRNA is broken down. psi inhibits basal glucose utilization in isolated rat soleus muscle with intact membrane with AD(50)42 mumol/l and Cmax 66%. Psi at concentrations found in renal failure patients inhibits both the insulin- and tolbutamide-stimulated glucose utilization. Its inhibitory activity is partially additive with diltiazem inhibition and nonadditive in the case of magnesium depletion. It is concluded that psi inhibits glucose utilization at the level of Ca modulation in the insulin regulatory cascade.

A C-nucleotide base pair: methylpseudouridine-directed incorporation of formycin triphosphate into RNA catalyzed by T7 RNA polymerase.

With templates containing 2'-deoxy-1-methylpseudouridine (dm psi), T7 RNA polymerase catalyzes the incorporation of either adenosine triphosphate (ATP) or formycin triphosphate (FTP) into a growing chain of RNA with the same efficiency as with templates containing thymidine (dT). In each case, the overall rate of synthesis of full-length products containing formycin is about one-tenth of the rate of synthesis of analogous products containing adenosine. Analysis of the products of abortive initiation shows that incorporation of FMP into the growing oligonucleotide by T7 RNA polymerase is more likely to lead to premature termination of transcription than is incorporation of AMP. Nevertheless, the results demonstrate that T7 RNA polymerase tolerates the formation of a C-nucleotide transcription complex in which the nucleoside bases on both the template and the incoming nucleotide are joined to the ribose by a carbon-carbon bond. This result increases the prospects for further expanding the genetic alphabet via incorporation of new base pairs with novel hydrogen-bonding schemes (Piccirilli et al., 1990).

Low molecular weight immunosuppressive factors found in elevated amounts in cancer ascitic fluids of mice. 2. 1-Methyladenosine isolated from cancer ascitic fluids enhances Listeria infection in mice.

The low molecular weight fraction (mol wt less than 1,000) of Ehrlich cancer ascitic fluid has been known to enhance Listeria infection in mice. Chemical characterization of the entities in this fraction revealed four purine and pyrimidine analogues, i.e. uric acid, uracil, pseudouridine and 1-methyladenosine (m1Ado). When the effect of each of these components was studied on Listeria infection in mice, only m1Ado markedly enhanced the infection and killed the mice within a short period. The optimal enhancement was obtained when m1Ado was given intravenously to mice 3-6 days before the infection at a concentration of between 1 and 100 micrograms/mouse. On the other hand, uric acid, uracil and pseudouridine failed to show such an enhancing effect. m1Ado inhibited macrophage accumulation in the peritoneal cavity of mice after an intraperitoneal injection of phytohemagglutinin. Although m1Ado did not show any inhibitory effect on the phagocytic and bactericidal activities of macrophages in vitro, peritoneal macrophages obtained from mice which received m1Ado 3 days ahead revealed impaired bactericidal activity, suggesting the migration of different cell populations from the bone marrow of m1Ado-receiving mice. The results may suggest that m1Ado is a major factor in tumor ascites causing, in small doses, an impairment of macrophage functioning as can be detected in tumor-bearing hosts.

Inhibition of UDP-N-acetylglucosamine pyrophosphorylase by uridine.

UDP-N-acetylglucosamine pyrophosphorylases (UTP: 2-acetamido-2-deoxy-alpha-D-glucose-1-phosphate uridylyltransferase, EC 2.7.7.23) from baker's yeast and Neurospora crassa IFO 6178 were inhibited by uridine which is the nucleoside moiety of UDP-GlcNAc. The inhibition was shown in both directions of pyrophosphorolysis and of synthesis of UDP-GlcNAc. Kinetic analysis revealed that uridine demonstrated a noncompetitive type of inhibition with UDP-GlcNAc and competitive inhibition with PPi. The Ki values for the baker's yeast enzyme were 1.8 mM for UDP-GlcNAc and 0.16 mM for PPi, and the values for the Neurospora enzyme were 1.1 mM for UDP-GlcNAc and 0.15 mM for PPi, respectively. Uridine did not bind irreversibly to the enzyme, as the activity was restored with dialysis. No other nucleosides caused inhibition of the enzyme activity except uridine. Some uridine derivatives, such as 5-hydroxyuridine, 5,6-dihydrouridine and pseudouridine, also inhibited the enzyme activity. But doexyuridine showed only slight inhibition, and 5'-UMP and orotidine caused no inhibition of the enzyme activity.