Synthesis and Biological Evaluation of Pyrimidine and Purine α-L-2′,3′-Dideoxy Nucleosides

Abstract

A series of α-L-2′,3′-dideoxy nucleosides was prepared as potential antiviral agents. The pyrimidine nucleosides were prepared by standard Vorbrüggen coupling reactions. The purine analogues were prepared by enzymatic transfer of the dideoxy sugar from a pyrimidine to a purine base. These compounds were inactive against HIV-1, HBV, HSV-1 and -2, VZV, and HCMV.     

Thrombopoietin-Increased DNA-PK-Dependent DNA Repair Limits Hematopoietic Stem and Progenitor Cell Mutagenesis in Response to DNA Damage

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Highlights? Mpl loss increases γ-irradiation-induced genomic instability in HSPCs ? TPO promotes DNA repair in vitro and in vivo in HSPCs ? TPO increases DNA-PK activity and NHEJ-mediated repair efficiency in HSPCs ? A single TPO injection before mouse TBI limits long-term HSC injury and mutagenesis     

Cloning and functional analysis of the dpm2 and dpm3 genes from Trichoderma reesei expressed in a Saccharomyces cerevisiae dpm1Δ mutant strain

In Trichoderma reesei, dolichyl phosphate mannose (dpm) synthase, a key enzyme in the O-glycosylation process, requires three proteins for full activity. In this study, the dpm2 and dpm3 genes coding for the DPMII and DPMIII subunits of T. reesei DPM synthase were cloned and functionally analyzed after expression in the Saccharomyces cerevisiae dpm1Δ [genotype (BY4743; his3Δ1; /leu2Δ0; lys2Δ0; /ura3Δ0; YPR183w::kanMX4] mutant. It was found that apart from the catalytic subunit DPMI, the DPMIII subunit is also essential to form an active DPM synthase in yeast. Additional expression of the DPMII protein, considered to be a regulatory subunit of DPM synthase, decreased the enzymatic activity. We also characterized S. cerevisiae strains expressing the dpm1, 2, 3 or dpm1, 3 genes and analyzed the consequences of dpm expression on protein O-glycosylation in vivo and on the cell wall composition.     

Light-induced responses of slow oscillatory neurons of the rat olivary pretectal nucleus

Background

The olivary pretectal nucleus (OPN) is a small midbrain structure responsible for pupil constriction in response to eye illumination. Previous electrophysiological studies have shown that OPN neurons code light intensity levels and therefore are called luminance detectors. Recently, we described an additional population of OPN neurons, characterized by a slow rhythmic pattern of action potentials in light-on conditions. Rhythmic patterns generated by these cells last for a period of approximately 2 minutes.

Methodology

To answer whether oscillatory OPN cells are light responsive and whether oscillatory activity depends on retinal afferents, we performed in vivo electrophysiology experiments on urethane anaesthetized Wistar rats. Extracellular recordings were combined with changes in light conditions (light-dark-light transitions), brief light stimulations of the contralateral eye (diverse illuminances) or intraocular injections of tetrodotoxin (TTX).

Conclusions

We found that oscillatory neurons were able to fire rhythmically in darkness and were responsive to eye illumination in a manner resembling that of luminance detectors. Their firing rate increased together with the strength of the light stimulation. In addition, during the train of light pulses, we observed two profiles of responses: oscillation-preserving and oscillation-disrupting, which occurred during low- and high-illuminance stimuli presentation respectively. Moreover, we have shown that contralateral retina inactivation eliminated oscillation and significantly reduced the firing rate of oscillatory cells. These results suggest that contralateral retinal innervation is crucial for the generation of an oscillatory pattern in addition to its role in driving responses to visual stimuli.     

Release of Metal Ions from Orthodontic Appliances: An In Vitro Study

In this paper, we report the results of an in vitro experiment on the release of metal ions from orthodontic appliances composed of alloys containing iron, chromium, nickel, silicon, and molybdenum into artificial saliva. The concentrations of magnesium, aluminum, silicon, phosphorus, sulfur, potassium, calcium, titanium, vanadium, manganese, iron, cobalt, copper, zinc, nickel, and chromium were significantly higher in artificial saliva in which metal brackets, bands, and wires used in orthodontics were incubated. In relation to the maximum acceptable concentrations of metal ions in drinking water and to recommended daily doses, two elements of concern were nickel (573 vs. 15 μg/l in the controls) and chromium (101 vs. 8 μg/l in the controls). Three ion release coefficients were defined: α, a dimensionless multiplication factor; β, the difference in concentrations (in micrograms per liter); and γ, the ion release coefficient (in percent). The elevated levels of metals in saliva are thought to occur by corrosion of the chemical elements in the alloys or welding materials. The concentrations of some groups of dissolved elements appear to be interrelated.     

Purine nucleoside synthesis, an efficient method employing nucleoside phosphorylases

An improved method for the enzymatic synthesis of purine nucleosides is described. Pyrimidine nucleosides were used as pentosyl donors and two phosphorylases were used as catalysts. One of the enzymes, either uridine phosphorylase (Urd Pase) or thymidine phosphorylase (dThd Pase), catalyzed the phosphorolysis of the pentosyl donor. The other enzyme, purine nucleoside phosphorylase (PN Pase), catalyzed the synthesis of the product nucleoside by utilizing the pentose 1-phosphate ester generated from the phosphorolysis of the pyrimidine nucleoside. Urd Pase, dThd Pase, and PN Pase were separated from each other in extracts of Escherichia coli by titration with calcium phosphate gel. Each enzyme was further purified by ion-exchange chromatography. Factors that affect the stability of these catalysts were studied. The pH optima for the stability of Urd Pase, dThd Pase, and PN Pase were 7.6, 6.5, and 7.4, respectively. The order of relative heat stability was Urd Pase greater than PN Pase greater than dThd Pase. The stability of each enzyme increased with increasing enzyme concentration. This dependence was strongest with dThd Pase and weakest with Urd Pase. Of the substrates tested, the most potent stabilizers of Urd Pase, dThd Pase, and PN Pase were uridine, 2'-deoxyribose 1-phosphate, and ribose 1-phosphate, respectively. Some general guidelines for optimization of yields are given. In a model reaction, optimal product formation was obtained at low phosphate concentrations. As examples of the efficiency of the method, the 2'-deoxyribonucleoside of 6-(dimethylamino)purine and the ribonucleoside of 2-amino-6-chloropurine were prepared in yields of 81 and 76%, respectively.     

Alterations in protein secretion caused by metabolic engineering of glycosylation pathways in fungi

Due to its natural properties, Trichoderma reesei is commonly used in industry-scale production of secretory proteins. Since almost all secreted proteins are O-glycosylated, modulation of the activity of enzymes of the O-glycosylation pathway are likely to affect protein production and secretion or change the glycosylation pattern of the secreted proteins, altering their stability and biological activity. Understanding how the activation of different components of the O-glycosylation pathway influences the glycosylation pattern of proteins and their production and secretion could help in elucidating the mechanism of the regulation of these processes and should facilitate creation of engineered microorganisms producing high amounts of useful proteins. In this review we focus on data concerning Trichoderma, but also present some background information allowing comparison with other fungal species.     

Information Flow Analysis of Interactome Networks

Recent studies of cellular networks have revealed modular organizations of genes and proteins. For example, in interactome networks, a module refers to a group of interacting proteins that form molecular complexes and/or biochemical pathways and together mediate a biological process. However, it is still poorly understood how biological information is transmitted between different modules. We have developed information flow analysis, a new computational approach that identifies proteins central to the transmission of biological information throughout the network. In the information flow analysis, we represent an interactome network as an electrical circuit, where interactions are modeled as resistors and proteins as interconnecting junctions. Construing the propagation of biological signals as flow of electrical current, our method calculates an information flow score for every protein. Unlike previous metrics of network centrality such as degree or betweenness that only consider topological features, our approach incorporates confidence scores of protein–protein interactions and automatically considers all possible paths in a network when evaluating the importance of each protein. We apply our method to the interactome networks of Saccharomyces cerevisiae and Caenorhabditis elegans. We find that the likelihood of observing lethality and pleiotropy when a protein is eliminated is positively correlated with the protein's information flow score. Even among proteins of low degree or low betweenness, high information scores serve as a strong predictor of loss-of-function lethality or pleiotropy. The correlation between information flow scores and phenotypes supports our hypothesis that the proteins of high information flow reside in central positions in interactome networks. We also show that the ranks of information flow scores are more consistent than that of betweenness when a large amount of noisy data is added to an interactome. Finally, we combine gene expression data with interaction data in C. elegans and construct an interactome network for muscle-specific genes. We find that genes that rank high in terms of information flow in the muscle interactome network but not in the entire network tend to play important roles in muscle function. This framework for studying tissue-specific networks by the information flow model can be applied to other tissues and other organisms as well.