Synthesis and evaluation of Janus type nucleosides as potential HCV NS5B polymerase inhibitors

The synthesis of new ribo and 2′-β-C-methyl ribo Janus type nucleosides J-AA, J-AG and J-AU is reported along with their ability to block HCV and HIV replication. Their toxicity was also assessed in Huh7, human lymphocytes, CEM and Vero cells.     

Relative transgene expression frequencies in homozygous versus hemizygous transgenic mice

We have used a simple binomial model of stochastic transgene inactivation at the level of the chromosome or transgene, rather than the cellular level, for the analysis of two mouse transgenic lines that show variegated patterns of expression. This predicts the percentages of cells that express one, both or neither alleles of the transgene in homozygotes from the observed percentages of cells, which express the transgene in hemizygotes. It adequately explained the relationship between the numbers of cells expressing the transgene in hemizygous and homozygous mosaic 21OH/LacZ mouse adrenals and mosaic BLG/7 mouse mammary glands. The binomial model also predicted that a small proportion of cells in mosaic mammary glands of BLG/7 homozygotes would express both BLG/7 alleles but published data indicated that all cells expressing the transgene showed monoallelic expression. Although it didn’t fit all of the BLG/7 data as precisely as a more complex model, which used several ad hoc assumptions to explain these results, the simple binomial model was able to explain the relationship in observed transgene expression frequencies between hemizygous and homozygous mosaic tissues for both 21OH/LacZ and BLG/7 mice. It may prove to be a useful general model for analysing other transgenic animals showing mosaic transgene expression.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

5,6-Dihydro-5-aza-2′-deoxycytidine potentiates the anti-HIV-1 activity of ribonucleotide reductase inhibitors

The nucleoside analog 5,6-dihydro-5-aza-2′-deoxycytidine (KP-1212) has been investigated as a first-in-class lethal mutagen of human immunodeficiency virus type-1 (HIV-1). Since a prodrug monotherapy did not reduce viral loads in Phase II clinical trials, we tested if ribonucleotide reductase inhibitors (RNRIs) combined with KP-1212 would improve antiviral activity. KP-1212 potentiated the activity of gemcitabine and resveratrol and simultaneously increased the viral mutant frequency. G-to-C mutations predominated with the KP-1212-resveratrol combination. These observations represent the first demonstration of a mild anti-HIV-1 mutagen potentiating the antiretroviral activity of RNRIs and encourage the clinical translation of enhanced viral mutagenesis in treating HIV-1 infection.     

A sphingosine 1-phosphate receptor 2 selective allosteric agonist

Molecular probe tool compounds for the Sphingosine 1-phosphate receptor 2 (S1PR2) are important for investigating the multiple biological processes in which the S1PR2 receptor has been implicated. Amongst these are NF-κB-mediated tumor cell survival and fibroblast chemotaxis to fibronectin. Here we report our efforts to identify selective chemical probes for S1PR2 and their characterization. We employed high throughput screening to identify two compounds which activate the S1PR2 receptor. SAR optimization led to compounds with high nanomolar potency. These compounds, XAX-162 and CYM-5520, are highly selective and do not activate other S1P receptors. Binding of CYM-5520 is not competitive with the antagonist JTE-013. Mutation of receptor residues responsible for binding to the zwitterionic headgroup of sphingosine 1-phosphate (S1P) abolishes S1P activation of the receptor, but not activation by CYM-5520. Competitive binding experiments with radiolabeled S1P demonstrate that CYM-5520 is an allosteric agonist and does not displace the native ligand. Computational modeling suggests that CYM-5520 binds lower in the orthosteric binding pocket, and that co-binding with S1P is energetically well tolerated. In summary, we have identified an allosteric S1PR2 selective agonist compound.     

Productivity differences among loblolly pine genotypes are independent of individual-tree biomass partitioning and growth efficiency

Genetic differences in individual-tree biomass partitioning, growth efficiency, and stem relative growth rate (RGR) could confer intraspecific productivity differences and might strongly influence forest ecosystem carbon storage. We examined the relationship between genotype productivity (stem volume), whole-tree biomass partitioning, growth efficiency (stem wood production per unit leaf area), and stem RGR among nine different loblolly pine (Pinus taeda L.) genotypes from three different genetic groups of contrasting inherent genetic homogeneity: three open-pollinated (half-sib) families, three mass-control pollinated (full-sib) families, and three clonal varieties. We hypothesized that genotype productivity would be positively associated with increased partitioning to stem wood relative to other plant parts, higher stem RGR, and enhanced growth efficiency. After 3 years under plantation conditions, genotypes showed significant differences in stem volume, percent stem wood, percent branch wood, and partitioning to fine roots, yet no differences in stem RGR or growth efficiency. Furthermore, genotypic differences in stem volume were independent of genotypic differences in biomass partitioning, and overall, we found no evidence to support the hypothesized relationships. Even so, the observed variation in biomass partitioning has implications for forest C sequestration as genotypes which partition more biomass to long-lived biomass pools such as stems, may sequester more C. Moreover, the lack of a genetic relationship between stem volume and belowground partitioning suggests that highly productive genotypes may be planted without compromising belowground C storage.     

Conjugation in Hyalophysa chattoni Bradbury (Apostomatida): An adaptation to a symbiotic life cycle

Hyalophysa chattoni, borne as an encysted phoront on a crustacean's exoskeleton, metamorphoses to the trophont during the host's premolt. After the molt within 15 min to 2 h conjugants with food vacuoles appear in the exuvium, swimming along with the trophonts. Starvation in other ciliates usually precedes conjugation, but food vacuoles in conjugants do not preclude starvation. Only after ingestion and dehydration of vacuoles ceases, does digestion of exuvial fluid begin. Conjugants resorb their feeding apparatus as they fuse. A single imperforate membrane from each partner forms the junction membrane. In a reproductive cyst conjugants divide synchronously, but now the junction membrane is interrupted by pores and channels. After the last division the daughters undergo meiosis – two meiotic divisions and one mitotic division yielding two prokarya as they simultaneously differentiate into tomites. After fertilization, pairs separate and the synkaryon divides once into a macronuclear anlage and a micronucleus. Exconjugants leave the cyst and seek a host. The parental macronucleus remains active until the phoront stage when the anlage develops. Owing to random association of micronuclei during meiosis, Hyalophysa's exconjugants are more genetically diverse than exconjugants from conventional patterns of conjugation.