HIM1, a new yeast Saccharomyces cerevisiae gene playing a role in control of spontaneous and induced mutagenesis

We have identified a new Saccharomyces cerevisiae gene, HIM1, mapped on the right arm of the chromosome IV (ORF YDR317w), mutations in which led to an increase in spontaneous mutation rate and elevated the frequencies of mutations, induced by UV-light, nitrous acid, ethylmethane sulfonate and methylmethane sulfonate. At the same time, him1 mutation did not result in the increase of the sensitivity to the lethal action of these DNA-damaging agents. We tested the induced mutagenesis in double mutants carrying him1 mutation and mutations in other repair genes: apn1, blocking base excision repair; rad2, rev3, and rad54, blocking three principal DNA repair pathways; pms1, blocking mismatch repair; hsm2 and hsm3 mutations, which lead to a mutator effect. Epistatic analysis showed a synergistic interaction of him1 with pms1, apn1, and rad2 mutations, and epistasis with the rev3, the rad54, the hsm2, and the hsm3. To elucidate the role of the HIM1 in control of spontaneous mutagenesis, we checked the repair of DNA mispaired bases in the him1 mutant and discovered that it was not altered in comparison to the wild-type strain. In our opinion, our results suggest that HIM1 gene participates in the control of processing of mutational intermediates appearing during error-prone bypass of DNA damage.     

Synthesis and antiviral evaluation of benzimidazoles, quinoxalines and indoles from dehydroabietic acid

Several heterocycles, such as benzimidazoles, quinoxalines and indoles incorporated into a hydrophenanthrene and naphthalene skeleton, were synthesised from two useful ortho-bromonitro precursors derived from dehydroabietic acid: methyl 12-bromo-13-nitro-deisopropyldehydroabietate and methyl 12-bromo-13,14-dinitro-deisopropyldehydroabietate. The new heterocycles were evaluated for their activity in vitro against several RNA and DNA viruses.     

Differentiation of Human Umbilical Cord Matrix Mesenchymal Stem Cells into Neural-Like Progenitor Cells and Maturation into an Oligodendroglial-Like Lineage

Mesenchymal stem cells (MSCs) are viewed as safe, readily available and promising adult stem cells, which are currently used in several clinical trials. Additionally, their soluble-factor secretion and multi-lineage differentiation capacities place MSCs in the forefront of stem cell types with expected near-future clinical applications. In the present work MSCs were isolated from the umbilical cord matrix (Wharton''s jelly) of human umbilical cord samples. The cells were thoroughly characterized and confirmed as bona-fide MSCs, presenting in vitro low generation time, high proliferative and colony-forming unit-fibroblast (CFU-F) capacity, typical MSC immunophenotype and osteogenic, chondrogenic and adipogenic differentiation capacity. The cells were additionally subjected to an oligodendroglial-oriented step-wise differentiation protocol in order to test their neural- and oligodendroglial-like differentiation capacity. The results confirmed the neural-like plasticity of MSCs, and suggested that the cells presented an oligodendroglial-like phenotype throughout the differentiation protocol, in several aspects sharing characteristics common to those of bona-fide oligodendrocyte precursor cells and differentiated oligodendrocytes.     

Glycan-binding profile of DC-like cells

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.     

The effect of implantation on scaffoldless three-dimensional engineered bone constructs

Our laboratory has previously developed scaffoldless engineered bone constructs (EBC). Bone marrow stromal cells (BMSC) were harvested from rat femur and cultured in medium that induced osteogenic differentiation. After reaching confluence, the monolayer of cells contracted around two constraint points forming a cylinder. EBCs were placed in small diameter (0.5905 × 0.0625 in.) or large diameter (0.5905 × 0.125 in.) silicone tubing and implanted intramuscularly in the hind limb of a rat. Bone mineral content (BMC) of the EBC was analyzed before implantation and at 1 and 2 mo following implantation and compared to that of native femur bone at different stages of development. Negligible BMC was observed in E-20 femur or EBCs prior to implantation. One-month implantation in both small and large tubing increased BMC in the EBC. BMC of EBC from large tubing was greater than in 14 d rat neonatal femurs, but was 2% and 3% of BMC content in adult bone after 1 and 2 mo of implantation, respectively. Alizarine Red and osteopontin staining of the EBCs before and after implantation confirmed increased bone mineralization in the implanted EBCs. Implanted EBCs also had extensive vascularization. Our data suggest that BMSC can be successfully used for the generation of scaffoldless EBC, and this model can be potentially used for the generation of autologous bone transplants in humans.