The role of receptor MAS in microglia-driven retinal vascular development.

OBJECTIVE: The receptor MAS, encoded by Mas1, is expressed in microglia and its activation has been linked to anti-inflammatory actions. However, microglia are involved in several different processes in the central nervous system, including the promotion of angiogenesis. We therefore hypothesized that the receptor MAS also plays a role in angiogenesis via microglia. APPROACH AND RESULTS: To assess the role of MAS on vascular network development, flat-mounted retinas from 3-day-old wild-type (WT) and Mas1-/- mice were subjected to Isolectin B4 staining. The progression of the vascular front was reduced (- 24%, p < 0.0001) and vascular density decreased (- 38%, p < 0.001) in Mas1-/- compared to WT mice with no change in the junction density. The number of filopodia and filopodia bursts were decreased in Mas1-/- mice at the vascular front (- 21%, p < 0.05; - 29%, p < 0.0001, respectively). This was associated with a decreased number of vascular loops and decreased microglial density at the vascular front in Mas1-/- mice (-32%, p < 0.001; - 26%, p < 0.05, respectively). As the front of the developing vasculature is characterized by reduced oxygen levels, we determined the expression of Mas1 following hypoxia in primary microglia from 3-day-old WT mice. Hypoxia induced a 14-fold increase of Mas1 mRNA expression (p < 0.01). Moreover, stimulation of primary microglia with a MAS agonist induced expression of Notch1 (+ 57%, p < 0.05), Dll4 (+ 220%, p < 0.001) and Jag1 (+ 137%, p < 0.001), genes previously described to mediate microglia/endothelial cell interaction during angiogenesis. CONCLUSIONS: Our study demonstrates that the activation of MAS is important for microglia recruitment and vascular growth in the developing retina.

Brain serotonin deficiency leads to social communication deficits in mice.

A deficit in brain serotonin is thought to be associated with deteriorated stress coping behaviour, affective disorders and exaggerated violence. We challenged this hypothesis in mice with a brain-specific serotonin depletion caused by a tryptophan hydroxylase 2 (TPH2) deficiency. We tested TPH2-deficient (Tph2(-/-)) animals in two social situations. As juveniles, Tph2(-/-) mice displayed reduced social contacts, whereas ultrasonic vocalizations (USVs) were unchanged within same-sex same-genotype pairings. Interestingly, juvenile females vocalized more than males across genotypes. Sexually naive adult males were exposed to fresh male or female urine, followed by an interaction with a conspecific, and re-exposed to urine. Although Tph2(-/-) mice showed normal sexual preference, they were hyper-aggressive towards their interaction partners and did not vocalize in response to sexual cues. These results highlight that central serotonin is essential for prosocial behaviour, especially USV production in adulthood, but not for sexual preference.

[[Length polymorphism of minisatellite repeat B2-VNTR of the bradykinin B2 receptor gene in healthy Russians and in patients with coronary heart disease].

Bradykinin B2 receptor is involved in many processes, including the regulation of blood pressure and smooth muscle contraction, vasodilation, inflammation, edema, cell proliferation, pain. It is suggested that this receptor may be one of the factors that have cardioprotective and infarct-limiting effects. It is assumed that certain genetic variants in both coding and non-coding regions ofBDKRB2 gene may influence its expression. In the 3'-untranslated region of BDKRB2 exon 3 the minisatellite repeat B2-VNTR is located. B2-VNTR has previously been shown to affect the BDKRB2 mRNA stability. Therefore, it is important to perform the molecular genetic analysis of this minisatellite in patients with different forms of coronary heart disease in order to reveal possible associations between specific B2-VNTR alleles and certain clinical forms of coronary heart disease. In the present study, a comparative analysis of the allele and genotype frequencies of B2-VNTR was carried out in groups of healthy individuals and patients with two clinical forms of coronary heart disease (angina pectoris and myocardial infarction), ethnically Russian. The results of the B2-VNTR length polymorphism analysis indicate that this tandem repeat may be attributed to a class of low polymorphic and non-hypervariable minisatellite. In all analyzed groups we revealed three B2-VNTR alleles, consisting of 43, 38 and 33 repeat units. Alleles of 43 and 33 repeats were major in all investigated groups. No statistically significant differences were found in the B2-VNTR allele and genotype frequencies between men and women in control group, and also between healthy men and men with angina pectoris and myocardial infarction. Thus, B2-VNTR length polymorphism was not associated with these clinical forms of coronary heart disease in Russian men. However, we do not exclude the possibility of association between the B2-VNTR short alleles (38 and 33 repeats) and cardioprotective effects of bradykinin B2 receptor in women with coronary heart disease. This hypothesis requires further investigation.

[Reprogramming of somatic cells. Problems and solutions].

An adult mammal is composed of more than 200 different types of specialized somatic cells whose differentiated state remains stable over the life of the organism. For a long time it was believed that the differentiation process is irreversible, and the transition between the two types of specialized cells is impossible. The possibility of direct conversion of one differentiated cell type to another was first shown in the 80s of the last century in experiments on the conversion of fibroblasts into myoblasts by ectopic expression of the transcription factor MyoD. Surprisingly, this technology has remained unclaimed in cell biology for a long time. Interest in it revived after 200 thanks to the research of Novel Prize winner Shinya Yamanaka who has shown that a small set of transcription factors (Oct4, Sox2, Klf4 and c-Myc) is capable of restoring pluripotency in somatic cells which they lost in the process of differentiation. In 2010, using a similar strategy and the tissue-specific transcription factors Vierbuchen and coauthors showed the possibility of direct conversion of fibroblasts into neurons, i. e. the possibility of transdifferentiation of one type of somatic cells in the other. The works of these authoras were a breakthrough in the field of cell biology and gave a powerful impulse to the development of cell technologies for the needs of regenerative medicine. The present review discusses the main historical discoveries that preceded this work, evaluates the status of the problem and the progress in the development of methods for reprogramming at the moment, describes the main approaches to solving the problems of reprogramming of somatic cells into neuronal, and briefly discusses the prospect of application of reprogramming and transdifferentiation of cells for such important application areas as regenerative medicine, cell replacement therapy and drug screening.

[Genetic manipulation and studying of differentiation properties of rat induced pluripotent stem cells].

Induced pluripotent stem (iPS) cells are derived from somatic cells reprogrammed to the pluripotent state by the induced expression of defined transcription factors, achieved for the first time by the seminal work of Takahashi and Yamanaka. This new type of pluripotent cells has offered new exciting options in regenerative medicine allowing the replacement of cells and organs with the patient's own cells thereby avoiding immunological complications. In order to develop such technologies in approved animal models, iPS cells were also generated from rodents. Of course, the most important model for studying of different diseases is rat. In this study, we present a method suitable for rat iPS cells genetic modification by stable transfection and show necessary conditions for the first stages of direct differentiation.

[Generation of rat induced pluripotent stem cells: the analysis of reprogramming and culturing media].

The rat represents very important, superior in many respects to the mous, animal model for studying pharmacology, physiology, ageing, cardiovascular etc. However, numerous attempts to derive rat ES cells necessary to carry out loss-of-gene-function studies have not been successful thus far. Therefore rat induct pluripotent stem cells (or riPS) should provide a notable alternative to ES cell, allowing to study gene functions in this valuable animal model. Here we report an improved lentivirus-based riPS derivation protocol that makes use of small inhibitors of MEK and GSK3. We show that the excision of proviruses does not affect neither karyotype and pluripotency state of these cells. Also, we propose genetic tool for an improvement of the quality of riPS cells in culture. These data may prompt further iPS-based gene targeting in rat as well as the development iPS-based gene therapies, using this animal model.

[CENTB5 gene expression in human and mouse].

Centaurin beta5 with unclear function belongs to protein family of centaurins. Human centaurin beta5 is encoded by gene CENTB5 whose intron 14-15 contains low variable minisatellite UPS29, and mouse homolog CENTB5 in analogous intron contains imperfect microsatellite repeat (CATG)19. Earlier we found the association between an occurrence of short UPS29 alleles with some forms of Parkinson disease and epilepsy. Besides this, both human and mice CENTB5 are localized in the same synteny group with SCNN1D and ACOT7 genes which are known to be expressed predominantly in nervous system. Mutations in these genes are connected with neurodegenerative processes and epilepsy. It is known that intra-intronic sequences can modulate genes of their location and neighbor and even remote genes. Using RT-PCR we carried out simultaneous analysis of CENTB5, SCNN1D and ACOT7 genes expression. Potential possibility of human intra-intronic tandem repeat UPS29 and of mouse intra-intronic tandem repeat (CATG)19 to regulate/modulate CENTB5, SCNN1D and ACOT7 activity was evaluated in silico. It was found that all these genes were expressed in all studied organs and tissues. It is suggested that minisatellite locus UPS29 can regulate an activity of CENTB5, SCNN1D and ACOT7 in nervous system cells.

Cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

Direct lineage conversion is a promising approach for disease modeling and regenerative medicine. Cell divisions play a key role in reprogramming of somatic cells to pluripotency, however their role in direct lineage conversion is not clear. Here we used transdifferentiation of fibroblasts into neuronal cells by forced expression of defined transcription factors as a model system to study the role of cellular division in the direct conversion process. We have shown that conversion occurs in the presence of the cell cycle inhibitors aphidicolin or mimosine. Moreover, overexpression of the cell cycle activator cMyc negatively influences the process of direct conversion. Overall, our results suggest that cell divisions are not essential for the direct conversion of fibroblasts into neuronal cells.

[Modification of the effect of high temperature on biochemical processes by perturbed geomagnetic field]. / Modifikatsiia vliianiia povyshennoi temperatury okruzhaiushchei sredy na biokhimicheskie protsessy s pomoshch'iu iskazhennogo geomagnitnogo polia.

The biochemical data on the influence of perturbed geomagnetic field on the hormonal-mediator regulation systems are presented. The possibility of biological effects modification under combined action of perturbed geomagnetic field and high temperature of environment is discussed.

Bovine satellite DNA induces heterochromatinization of host chromosomal DNA in cells of trassatellite mouse embryonal carcinoma.

Embryonal teratocarcinoma F9 cells were transfected with a fragment (3.8 kb) of bovine satellite DNA IV (Sat), which is not homologous to mouse satellite DNA. FISH analysis revealed various chromosomal integration sites of integrated Sat in different transsatellite clones. After several passages, transsatellite had a tendency to spread along chromosome bearing Sat in one of the studied lines. The integrated transsatellites were enriched with prolonged single-strand DNA regions (SSR) revealed by FISH without previous chromosomal denaturation, and were unmethylated. The observed SSR are presumably supposed to represent intermediates of transsatellite DNA instability via unequal sister chromatid exchanges. DAPI staining demonstrated that the integrated Sat induced the formation of prominent ectopic neoheterochromatin blocks in regions adjacent to integrated Sat. These blocks were located exclusively between integrated Sat and centromeric heterochromatin. Thus, mouse repetitive centromeric DNA (AT-rich, DAPI-positive) "spreads" along the chromosome in response to integration of the bovine satellite GC-rich DNA. The results obtained are discussed in the context of possible position effect variegation mechanisms operating in undifferentiated cells.

Exaggerated aggression and decreased anxiety in mice deficient in brain serotonin.

Serotonin is a major neurotransmitter in the central nervous system (CNS). Dysregulation of serotonin transmission in the CNS is reported to be related to different psychiatric disorders in humans including depression, impulsive aggression and anxiety disorders. The most frequently prescribed antidepressants and anxiolytics target the serotonergic system. However, these drugs are not effective in 20-30% of cases. The causes of this failure as well as the molecular mechanisms involved in the origin of psychological disorders are poorly understood. Biosynthesis of serotonin in the CNS is initiated by tryptophan hydroxylase 2 (TPH2). In this study, we used Tph2-deficient (Tph2(-/-)) mice to evaluate the impact of serotonin depletion in the brain on mouse behavior. Tph2(-/-) mice exhibited increased depression-like behavior in the forced swim test but not in the tail suspension test. In addition, they showed decreased anxiety-like behavior in three different paradigms: elevated plus maze, marble burying and novelty-suppressed feeding tests. These phenotypes were accompanied by strong aggressiveness observed in the resident-intruder paradigm. Despite carrying only one copy of the gene, heterozygous Tph2(+/-) mice showed only 10% reduction in brain serotonin, which was not sufficient to modulate behavior in the tested paradigms. Our findings provide unequivocal evidence on the pivotal role of central serotonin in anxiety and aggression.