Purification and characterization of uridine and thymidine phosphorylase from Lactobacillus casei

Uridine and thymidine phosphorylases have been purified to homogeneity from crude extracts of Lactobacillus casei. Both enzymes had an apparent molecular mass of about 80 kDa. Uridine phosphorylase consisted of four identical subunits while thymidine phosphorylase was composed of two identical ones. The sequence of 23 amino-acid residues from its N-terminal end was analyzed. Uridine phosphorylase had a Km of 5.0 x 10(-3) M for uridine and 1.24 x 10(-1) M for phosphate, while thymidine phosphorylase had a Km of 1.32 x 10(-1) M for thymidine and 1.0 x 10(-1) M for phosphate. Uridine phosphorylase was equally active with uridine and 5-methyluridine, but had a low activity towards thymidine. Its activity was inhibited competitively by 3-O-methyl-alpha D-glucopyranoside, on the other hand thymidine phosphorylase activity was not affected by this compound. Thymidine phosphorylase showed specificity towards the deoxyribosyl moiety of the substrate. In addition, it required a nonsubstituted pyrimidine moiety or one which was substituted in position 5. The pattern of the double-reciprocal plots of the initial velocities vs. the concentrations of either one of the substrates, and the product inhibition kinetics, indicated that the catalytic mechanism of both enzymatic reactions is sequential rather than Ping-Pong and that the sequence of the addition of the substrates is random (rapid equilibrium). In the case of the uridine phosphorylase-catalyzed reaction, the products are also released randomly, while in the thymidine phosphorylase-catalyzed reaction deoxyribose 1-phosphate is released after thymine.     

Synthesis of daunosamine-containing disaccharides

Treatment of 2,3,6-trideoxy-1,4-di-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-l-lyxo-hexopyranose (1) with benzyl 2,3-dideoxy-d-glycero-pentopyranoside and p-toluenesulfonic acid gave a mixture of benzyl 2,3,6-trideoxy-4-O-p-nitrobenzoyl-3- (trifluoroacetamido)-l-lyxo-hexopyranoside (49%) and benzyl 2,3-dideoxy-4-O-[2,3,6-trideoxy-4-O-(p-nitrobenzoyl)-3-(trifluoroacetamido)-α-l-lyxo-hexopyranosyl]-d-glycero-pentopyranoside (4, 20 %). The structure of the disaccharide 4 was confirmed by a detailed, mass-spectrometric analysis in three modes, namely, negative- and positive-ion, chemical ionization, and electron impact. Similar treatment of the bis(p-nitrobenzoate) 1 with ethyl 2,3-dideoxy-d-glycero-pentopyranoside gave the ethyl glycoside and the desired disaccharide, showing that the transglycosylation is not restricted to benzyl glycosides. Removal of the p-nitrobenzoyl and the benzyl groups from 4 gave the disaccharide 2,3-dideoxy-4-O-(2,3,6-trideoxy-3-trifluoroacetamido-α-l-lyxo-hexopyranosyl)-d-glycero-pentopyranose.     

Epigenetic Alterations at Genomic Loci Modified by Gene Targeting in Arabidopsis thaliana

Gene Targeting (GT) is the integration of an introduced vector into a specific chromosomal site, via homologous recombination. It is considered an effective tool for precise genome editing, with far-reaching implications in biological research and biotechnology, and is widely used in mice, with the potential of becoming routine in many species. Nevertheless, the epigenetic status of the targeted allele remains largely unexplored. Using GT-modified lines of the model plant Arabidopsis thaliana, we show that the DNA methylation profile of the targeted locus is changed following GT. This effect is non-directional as methylation can be either completely lost, maintained with minor alterations or show instability in the generations subsequent to GT. As DNA methylation is known to be involved in several cellular processes, GT-related alterations may result in unexpected or even unnoticed perturbations. Our analysis shows that GT may be used as a new tool for generating epialleles, for example, to study the role of gene body methylation. In addition, the analysis of DNA methylation at the targeted locus may be utilized to investigate the mechanism of GT, many aspects of which are still unknown.     

Collective Motion of Spherical Bacteria

A large variety of motile bacterial species exhibit collective motions while inhabiting liquids or colonizing surfaces. These collective motions are often characterized by coherent dynamic clusters, where hundreds of cells move in correlated whirls and jets. Previously, all species that were known to form such motion had a rod-shaped structure, which enhances the order through steric and hydrodynamic interactions. Here we show that the spherical motile bacteria Serratia marcescens exhibit robust collective dynamics and correlated coherent motion while grown in suspensions. As cells migrate to the upper surface of a drop, they form a monolayer, and move collectively in whirls and jets. At all concentrations, the distribution of the bacterial speed was approximately Rayleigh with an average that depends on concentration in a non-monotonic way. Other dynamical parameters such as vorticity and correlation functions are also analyzed and compared to rod-shaped bacteria from the same strain. Our results demonstrate that self-propelled spherical objects do form complex ordered collective motion. This opens a door for a new perspective on the role of cell aspect ratio and alignment of cells with regards to collective motion in nature.     

Spatial and Temporal Biogeography of Soil Microbial Communities in Arid and Semiarid Regions

Microbial communities in soils may change in accordance with distance, season, climate, soil texture and other environmental parameters. Microbial diversity patterns have been extensively surveyed in temperate regions, but few such studies attempted to address them with respect to spatial and temporal scales and their correlations to environmental factors, especially in arid ecosystems. In order to fill this gap on a regional scale, the molecular fingerprints and abundance of three taxonomic groups – Bacteria, α-Proteobacteria and Actinobacteria – were sampled from soils 0.5–100 km apart in arid, semi-arid, dry Mediterranean and shoreline Mediterranean regions in Israel. Additionally, on a local scale, the molecular fingerprints of three taxonomic groups – Bacteria, Archaea and Fungi – were sampled from soils 1 cm–500 m apart in the semi-arid region, in both summer and winter. Fingerprints of the Bacteria differentiated between all regions (P<0.02), while those of the α-Proteobacteria differentiated between some of the regions (0.01<P<0.09), and actinobacterial fingerprints were similar among all regions (P>0.05). Locally, fingerprints of archaea and fungi did not display distance-decay relationships (P>0.13), that is, the dissimilarity between communities did not increase with geographic distance. Neither was this phenomenon evident in bacterial samples in summer (P>0.24); in winter, however, differences between bacterial communities significantly increased as the geographic distances between them grew (P<0.01). Microbial community structures, as well as microbial abundance, were both significantly correlated to precipitation and soil characteristics: texture, organic matter and water content (R²>0.60, P<0.01). We conclude that on the whole, microbial biogeography in arid and semi-arid soils in Israel is determined more by specific environmental factors than geographic distances and spatial distribution patterns.     

Physico-chemical foundations underpinning microarray and next-generation sequencing experiments

Hybridization of nucleic acids on solid surfaces is a key process involved in high-throughput technologies such as microarrays and, in some cases, next-generation sequencing (NGS). A physical understanding of the hybridization process helps to determine the accuracy of these technologies. The goal of a widespread research program is to develop reliable transformations between the raw signals reported by the technologies and individual molecular concentrations from an ensemble of nucleic acids. This research has inputs from many areas, from bioinformatics and biostatistics, to theoretical and experimental biochemistry and biophysics, to computer simulations. A group of leading researchers met in Ploen Germany in 2011 to discuss present knowledge and limitations of our physico-chemical understanding of high-throughput nucleic acid technologies. This meeting inspired us to write this summary, which provides an overview of the state-of-the-art approaches based on physico-chemical foundation to modeling of the nucleic acids hybridization process on solid surfaces. In addition, practical application of current knowledge is emphasized.     

Therapy model for advanced intracerebral B16 mouse melanoma using radiation therapy combined with immunotherapy

A reproducible therapy model for advanced intracerebral B16 melanoma is reported. Implanted tumors (D0), suppressed by a single 15 Gy radiosurgical dose of 100 kVp X-rays (D8), were further suppressed by a single ip injection of a Treg-depleting mAb given 2 days prior to the initiation (D9) of four weekly then eight bi-monthly sc injections of GMCSF-transfected, mitotically disabled B16 cells. The trends of seven independent experiments were similar to the combined result: The median (days) [SD/total N] of survival went from 15[1.09/62] (no treatment control) to 35.8[8.8/58] (radiation therapy only) to 52.5[13.5/57] (radiation therapy plus immunotherapy). Within 2 weeks after immunization, tumors in mice receiving radiation therapy plus immunotherapy were significantly smaller than tumors in mice treated only with radiosurgery. Splenocytes and lymph node cells from immunized mice showed increased interferon γ production when cultured with syngeneic tumor cells. We suggest that our model will be useful for the development and testing of novel combination therapies for brain tumors.