Production of endothelial progenitor cells obtained from human Wharton's jelly using different culture conditions

Endothelial progenitor cells (EPC) participate in revascularization and angiogenesis. EPC can be cultured in vitro from mononuclear cells of peripheral blood, umbilical cord blood or bone marrow; they also can be transdifferentiated from mesenchymal stem cells (MSC). We isolated EPCs from Wharton's jelly (WJ) using two methods. The first method was by obtaining MSC from WJ and characterizing them by flow cytometry and their adipogenic and osteogenic differentiation, then applying endothelial growth differentiating media. The second method was by direct culture of cells derived from WJ into endothelial differentiating media. EPCs were characterized by morphology, Dil-LDL uptake/UEA-1 immunostaining and testing the expression of endothelial markers by flow cytometry and RT-PCR. We found that MSC derived from WJ differentiated into endothelial-like cells using simple culture conditions with endothelium induction agents in the medium.     

A large proportion of mediastinal and perirenal visceral fat of Siberian adult people is formed by UCP1 immunoreactive multilocular and paucilocular adipocytes

Journal of Physiology and Biochemistry - Many deleterious consequences for health of excessive fat accumulation are due to visceral fat. Browning of visceral fat is mainly cold dependent and has...     

Evolution of spore release mechanisms in the saprolegniaceae (Oomycetes): evidence from a phylogenetic analysis of internal transcribed spacer sequences

Classical studies on spore release within the Saprolegniaceae (Oomycetes) led to the proposition that different mechanisms of sporangial emptying represent steps in an evolutionary transition series. We have reevaluated this idea in a phylogenetic framework using internal transcribed spacer sequences of four genera. These data were compared with the response to osmotic stress exhibited by each taxon. Saprolegnia emerges as the most basal genus, sister to Achlya, Thraustotheca, and Dictyuchus. Achlya and Thraustotheca are most closely related, while Dictyuchus appears to have evolved along a separate evolutionary lineage. The resulting phylogenetic framework is consistent with the idea that the mechanism of sporangial emptying exhibited by Saprolegnia represents the plesiomorphic condition from which the other mechanisms were derived independently. These alternative mechanisms of spore release may have resulted from a small number of mutations that inhibited axonemal development and altered the temporal and spatial expression of lytic enzymes that degrade the sporangial wall. Copyright 1998 Academic Press.     

Isolation and characterization of high affinity aptamers against DNA polymerase iota

Human DNA-polymerase iota (Pol ι) is an extremely error-prone enzyme and the fidelity depends on the sequence context of the template. Using the in vitro systematic evolution of ligands by exponential enrichment (SELEX) procedure, we obtained an oligoribonucleotide with a high affinity to human Pol ι, named aptamer IKL5. We determined its dissociation constant with homogenous preparation of Pol ι and predicted its putative secondary structure. The aptamer IKL5 specifically inhibits DNA-polymerase activity of the purified enzyme Pol ι, but did not inhibit the DNA-polymerase activities of human DNA polymerases beta and kappa. IKL5 suppressed the error-prone DNA-polymerase activity of Pol ι also in cellular extracts of the tumor cell line SKOV-3. The aptamer IKL5 is useful for studies of the biological role of Pol ι and as a potential drug to suppress the increase of the activity of this enzyme in malignant cells.     

Comparative genetic analysis of the hexaploid wheat Triticum petropavlovskyi Udasz. et Migusch. and Triticum aestivum L

A poorly studied species of hexaploid wheat Triticum petropavlovskyi Udacz. et Migusch. was compared with common wheat Triticum aestivum L. by means of monosomic and genetic analyses of F2 hybrids. Triticum petropavlovskyi was found to carry 13 dominant genes determining its morphological and physiological characters and regular bivalent conjugation of chromosomes. These genes were allelic to the respective genes of common wheat and were located in the same chromosomes. The modes of gene interaction were also the same. There was simple dominance for most genes studied and complementary interaction for the genes of hybrid dwarfism and hybrid necrosis. Triticum petropavlovskyi had the following dominant genes: Hg (downy glume); Rg1 (red glume color); Hl (downy leaf); Hn (downy node); Pa (pubescent auricles); Q (speltlike ears); D1 (grass-clump dwarfism); Ne1 (hybrid necrosis); Ph1 and Ph2 (genes of bivalent conjugation preventing homoeologous chromosomes from pairing); and Vrn1, Vrn2, Vrn3, and Vrn4 (genes of the spring habit). The gene Vrn1, which caused an increase in ear emergence time and a pronounced response to vernalization, was poorly expressed. T. petropavlovskyi was earlier demonstrated to have a species-specific gene P or Eg (elongated glume), which was not allelic to the gene Eg of the tetraploid T. polonicum L. The data obtained indicate that T. petropavlovskyi has originated from T. aestivum via mutations.     

Identification of a new <Emphasis Type="Italic">Vrn</Emphasis>-<Emphasis Type="Italic">B1</Emphasis> allele using two near-isogenic wheat lines with difference in heading time

We conducted a molecular analysis of the Vrn-B1 gene in two near-isogenic lines (NILs) carrying the dominant Vrn-B1 ^S and Vrn-B1 ^Dm alleles from the Saratovskaya 29 and Diamant 2 cultivars, respectively. These lines are characterized by different times of ear emergence. PCR analysis and subsequent sequencing of the regulatory regions of Vrn-B1 revealed the full identity of the promoter region in both alleles. Simultaneously, we found significant differences in the structure of the first intron of the Vrn-B1 ^S allele when compared to Vrn-B1 ^Dm ; specifically, the deletion of 0.8 kb coupled with the duplication of 0.4 kb. We suggest that these changes in intron 1 of Vrn-B1 ^S caused earlier ear emergence in the corresponding NIL. The unusual structure of intron 1 within the Vrn-B1 ^S allele was described for the first time in this study. The allele Vrn-B1 ^Dm was almost identical with the previously studied sequence of the Vrn-B1a allele of T. aestivum, Triple Dirk B. We designated the new Vrn-B1 ^S allele as Vrn-B1c. PCR analysis of the Vrn-B1 gene in 26 spring wheat cultivars of both Russian and foreign breeding revealed that 16 of them contain the Vrn-B1a allele and 6 contain the Vrn-B1c allele. Other cultivars studied contained the recessive vrn-B1 gene, except for Novosibirskaya 67. This study demonstrates that the traditional system of Vrn-1 markers does not fully encompass the allelic diversity of these genes because none of the cultivars containing the Vrn-B1c allele gave a PCR product using the previously developed set of primers for identification of the Vrn-B1 locus. We showed that the newly characterized Vrn-B1c allele is widely distributed among different genotypes of spring wheat. The findings indicate the impact of structural changes in the first intron of Vrn-1 on the vernalization response and heading time.     

Effect of coculturing canine notochordal,nucleus pulposus and mesenchymal stromal cells for intervertebral disc regeneration

IntroductionEarly degenerative changes in the nucleus pulposus (NP) are observed after the disappearance of notochordal cells (NCs). Thus, it has been suggested that NCs play an important role in maintaining the NP and may have a regenerative potential on other cells of the NP. As the number of resident NP cells (NPCs) decreases in a degenerating disc, mesenchymal stromal (stem) cells (MSCs) may be used for cell supplementation. In this study, using cells of one species, the regenerative potential of canine NCs was assessed in long-term three-dimensional coculture with canine NPCs or MSCs.MethodsCanine NCs and canine NPCs or MSCs were cocultured in alginate beads for 28 days under hypoxic and high-osmolarity conditions. Cell viability, cell morphology and DNA content, extracellular matrix production and expression of genes related to NC markers (Brachyury, KRT18) and NP matrix production (ACAN, COL2A1, COL1A1) were assessed after 1, 15 and 28 days of culture.ResultsNCs did not completely maintain their phenotype (morphology, matrix production, gene expression) during 28 days of culture. In cocultures of NPCs and NCs, both extracellular matrix content and anabolic gene expression remained unchanged compared with monoculture groups, whereas cocultures of MSCs and NCs showed increased glycosaminoglycan/DNA. However, the deposition of these proteoglycans was observed near the NCs and not the MSCs. Brachyury expression in the MSC and NC coculture group increased in time. The latter two findings indicate a trophic effect of MSCs on NCs rather than vice versa.ConclusionsNo regenerative potential of canine NCs on canine NPCs or MSCs was observed in this study. However, significant changes in NC phenotype in long-term culture may have resulted in a suboptimal regenerative potential of these NCs. In this respect, NC-conditioned medium may be better than coculture for future studies of the regenerative potential of NCs.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0569-6) contains supplementary material, which is available to authorized users.     

Marker-Dependent Recombination in T4 Bacteriophage. I. Outline of the Phenomenon and Evidence Suggesting a Mismatch Repair Mechanism

In standard crosses, some rIIB mutants of T4 phage were found to be susceptible to an extra recombination mechanism to which the other mutants were much less susceptible. The following observations were interpreted as evidence for the mismatch-repair nature of the phenomenon: (1) Marker-dependent recombination generates exclusively double exchanges at both sides of the marker. (2) Marker-dependent recombination is highly sensitive to DNA base sequence at the site of the marker and to that at the corresponding site on the chromosome of the other parent. (3) Within certain limits, the contribution of the marker-dependent mechanism to the total recombination frequency is distance-independent and thus constitutes a constant component.