The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.     

Tandem affinity purification of functional TAP-tagged proteins from human cells

Tandem affinity purification (TAP) is a generic two-step affinity purification protocol for isolation of TAP-tagged proteins together with associated proteins. We used bacterial artificial chromosome to heterologously express TAP-tagged murine Sgo1 protein in human HeLa cells. This allowed us to test the functionality of the Sgo1-TAP protein by RNA interference-mediated depletion of the endogenous human Sgo1. Here, we present an optimized protocol for purification of TAP-tagged Sgo1 protein as well as KIAA1387 from HeLa cells with detailed instructions. The purification protocol can be completed in 1 day and it should be applicable to other proteins.     

In-Vivo Imaging of Cell Migration Using Contrast Enhanced MRI and SVM Based Post-Processing

The migration of cells within a living organism can be observed with magnetic resonance imaging (MRI) in combination with iron oxide nanoparticles as an intracellular contrast agent. This method, however, suffers from low sensitivity and specificty. Here, we developed a quantitative non-invasive in-vivo cell localization method using contrast enhanced multiparametric MRI and support vector machines (SVM) based post-processing. Imaging phantoms consisting of agarose with compartments containing different concentrations of cancer cells labeled with iron oxide nanoparticles were used to train and evaluate the SVM for cell localization. From the magnitude and phase data acquired with a series of T2*-weighted gradient-echo scans at different echo-times, we extracted features that are characteristic for the presence of superparamagnetic nanoparticles, in particular hyper- and hypointensities, relaxation rates, short-range phase perturbations, and perturbation dynamics. High detection quality was achieved by SVM analysis of the multiparametric feature-space. The in-vivo applicability was validated in animal studies. The SVM detected the presence of iron oxide nanoparticles in the imaging phantoms with high specificity and sensitivity with a detection limit of 30 labeled cells per mm³, corresponding to 19 μM of iron oxide. As proof-of-concept, we applied the method to follow the migration of labeled cancer cells injected in rats. The combination of iron oxide labeled cells, multiparametric MRI and a SVM based post processing provides high spatial resolution, specificity, and sensitivity, and is therefore suitable for non-invasive in-vivo cell detection and cell migration studies over prolonged time periods.     

The application of the RT-PCR method for the staging of the prostate cancer progression

Molecular biology seems to bring more convincing markers for the detection of prostate cancer as well as the development of metastases than immunohistochemistry. The main goal of present work was to detect the expression of prostate specific antigen (PSA) and prostate-specific membrane antigen (PSM) genes in the micrometastases by the RT-PCR to assess the progression of prostate cancer. We analyzed 50 patients: 28 patients with clinically localized or locally advanced prostate cancer who underwent radical prostatectomy, 7 patients with clinically proven metastases, 8 patients with benign prostatic hyperplasia, and 7 healthy young men. The results of RT-PCR in the first group of 28 patients varied, however, they were in good correlation with the health status of the patients. Positive results of PSA and notably for PSM were good predictors of beginning metastasing process. Seven patients with metastatic disease had positive RT-PCR results both for PSA and PSM. All of the patients with benign prostatic hyperplasia and healthy young men had negative RT-PCR results for PSA and PSM. The study showed that positive RT-PCR results for PSA and especially for PSM correlated well with the progression of the disease and negative results reflected good health status of the patients.     

Grass cover on forest clear-cut areas ameliorates some soil chemical properties

Clear-cut areas formed after forest decline due to acid deposition, pest attacks, or wind-breaks in temperate mountainous regions are often populated by grass (mainly Calamagrostis villosa). This study focused on the changes of soil chemical characteristics under the grass cover replacing the forest, focusing mainly on aluminium (Al) speciation. Clear-cut area due to strong acid deposition in the Jizera Mountains (Northern Bohemia) was studied. The soils under grass cover exhibit higher pH values and lower exchangeable Al content compared to adjacent surviving forest. Mobile Al species under the grass have larger proportion of non-toxic organic complexes. The content of exchangeable base cations is slightly higher under the grass. The positive effect of grass on soil chemistry was enhanced by liming. The temporary grass cover can therefore improve soil chemical quality for following reforestation. However, the differences are generally limited to surface organic horizons. Similar results were found also on a bark-beetle clear-cut area in the Bohemian Forest (Southern Bohemia) with smaller acid deposition; nevertheless, most differences were not significant there.     

phiGENOME: an integrative navigation throughout bacteriophage genomes

phiGENOME is a web-based genome browser generating dynamic and interactive graphical representation of phage genomes stored in the phiSITE, database of gene regulation in bacteriophages. phiGENOME is an integral part of the phiSITE web portal (http://www.phisite.org/phigenome) and it was optimised for visualisation of phage genomes with the emphasis on the gene regulatory elements. phiGENOME consists of three components: (i) genome map viewer built using Adobe Flash technology, providing dynamic and interactive graphical display of phage genomes; (ii) sequence browser based on precisely formatted HTML tags, providing detailed exploration of genome features on the sequence level and (iii) regulation illustrator, based on Scalable Vector Graphics (SVG) and designed for graphical representation of gene regulations. Bringing 542 complete genome sequences accompanied with their rich annotations and references, makes phiGENOME a unique information resource in the field of phage genomics.     

Simulation of intrafollicular conditions prevents GVBD in bovine oocytes: a better alternative to affect their developmental capacity after two-step culture

To increase the developmental competence of bovine oocytes isolated from small, medium, and large follicles (2-3, 3-4, and 4-6 mm in diameter, respectively), we tried to modify the conditions for their in vitro culture. The first step involved conditions maintaining at least for 48 hr a reversible inhibition of the germinal vesicle breakdown (GVBD) and the second step stimulated the resumption of meiosis and completion of nuclear and cytoplasmic maturation during the subsequent 20-22 hr of culture. The effectiveness of this model depended mainly on the medium composition (reduced NaHCO3, substitution of serum with serum albumin, addition of antioxidants (curcumin), increased viscosity by agar, the reduction of oxygen concentration (within 6%-8%), the reduction of the proportion between the number of cumulus-oocyte complexes (COCs), and the reduction of the amount of a medium (within 6-7 mul per COC) to amplify the GVBD-inhibitory effect of oocyte surrounding granulosa cells. The COCs were incubated in clumps of 6-7 COCs. The effectiveness and reversibility of GVBD inhibition depended also on the duration of COCs isolation. The full reversibility of the GV block was controlled morphologically and also by measuring histone H1 and MAP kinase activities. The two-step versus one-step (24 hr) maturation technique was evaluated by the percentage of total and hatched blastocysts at day 9. When compared with one-step maturation, the two-step culture showed a slightly increased proportion of total and hatched blastocysts developed from growing follicles, mainly from the smallest category (13.9% vs. 7.1% and 9.2% vs. 3.3% for total blastocysts and hatched, respectively). However, the two-step culture of oocytes from large regressing follicles substantially reduced the blastocyst yield (9.7% vs. 39.1% and 4.9% vs. 26.7% for total blastocysts and hatched, respectively). The transfer of ten blastocysts (developed after two-step culture) to ten recipients resulted in seven pregnancies.     

The role of BRCA2 in replication-coupled DNA interstrand cross-link repair in vitro

Using a defined substrate DNA with a single psoralen interstrand cross-link (ICL), we studied the molecular mechanism of human ICL repair. In vitro ICL repair by human extracts is dependent on replication and is a largely error-free process. Extracts from a human BRCA2-defective mutant cell line, CAPAN-1, are severely compromised in ICL repair. Specifically, 'unhooked' but not fully repaired products accumulate in the reaction with CAPAN-1, and transient expression of BRCA2 in CAPAN-1 restores repair activity. Together, these results reveal that BRCA2 participates in repair of replication-mediated double-strand breaks generated when replication forks encounter ICLs. We also show that nucleotide excision repair is essential for the removal of the lesion left behind on one strand after unhooking. This study provides new mechanistic insights into the repair of ICLs in human cells.     

Microbial and chemical characterization of underwater fresh water springs in the Dead Sea

Due to its extreme salinity and high Mg concentration the Dead Sea is characterized by a very low density of cells most of which are Archaea. We discovered several underwater fresh to brackish water springs in the Dead Sea harboring dense microbial communities. We provide the first characterization of these communities, discuss their possible origin, hydrochemical environment, energetic resources and the putative biogeochemical pathways they are mediating. Pyrosequencing of the 16S rRNA gene and community fingerprinting methods showed that the spring community originates from the Dead Sea sediments and not from the aquifer. Furthermore, it suggested that there is a dense Archaeal community in the shoreline pore water of the lake. Sequences of bacterial sulfate reducers, nitrifiers iron oxidizers and iron reducers were identified as well. Analysis of white and green biofilms suggested that sulfide oxidation through chemolitotrophy and phototrophy is highly significant. Hyperspectral analysis showed a tight association between abundant green sulfur bacteria and cyanobacteria in the green biofilms. Together, our findings show that the Dead Sea floor harbors diverse microbial communities, part of which is not known from other hypersaline environments. Analysis of the water's chemistry shows evidence of microbial activity along the path and suggests that the springs supply nitrogen, phosphorus and organic matter to the microbial communities in the Dead Sea. The underwater springs are a newly recognized water source for the Dead Sea. Their input of microorganisms and nutrients needs to be considered in the assessment of possible impact of dilution events of the lake surface waters, such as those that will occur in the future due to the intended establishment of the Red Sea-Dead Sea water conduit.