Radiation mapping as one of main methods of creating high resolution maps of human and animal genomes

Radiation hybrid mapping (RH mapping) is considered as one of the main methods of constructing physical maps of mammalian genomes. In introduction, theoretical prerequisites of developing of the RH mapping and statistical methods of data analysis are discussed. Comparative characteristics of universal commercial panels of the radiation hybrid somatic cells (RH panels) are shown. In experimental part of the work, RH mapping is used to localise nucleotide sequences adjacent to NotI sites of human chromosome 3 with the aim to integrate contig map of NotI clones to comprehensive maps of human genome. Five nucleotide sequences adjacent to the sites of integration of papilloma virus in human genome and expressed in the cells of cervical cancer were localised. It was demonstrated that the region 13q14.3-q21.1 was enriched with nucleotide sequences involved in the processes of oncogenesis. RH mapping can be considered as one of the most perspective applications of the modern radiation biology in the field of molecular genetics, that is, in constructing physical maps of mammalian genomes with high resolution level.     

Targeted deletion of the integrin beta4 signaling domain suppresses laminin-5-dependent nuclear entry of mitogen-activated protein kinases and NF-kappaB, causing defects in epidermal growth and migration

The alpha6beta4 integrin-a laminin-5 receptor-mediates assembly of hemidesmosomes and recruitment of Shc and phosphoinositide 3-kinase through the unique cytoplasmic extension of beta4. Mice carrying a targeted deletion of the signaling domain of beta4 develop normally and do not display signs of skin fragility. The epidermis of these mice contains well-structured hemidesmosomes and adheres stably to the basement membrane. However, it is hypoplastic due to reduced proliferation of basal keratinocytes and undergoes wound repair at a reduced rate. Keratinocytes from beta4 mutant mice undergo extensive spreading but fail to proliferate and migrate in response to epidermal growth factor (EGF) on laminin-5. EGF causes significant phosphorylation of extracellular signal-regulated kinase (ERK) and Jun N-terminal protein kinase (JNK) and phosphorylation and degradation of IkappaB in beta4 mutant cells adhering to laminin-5. Unexpectedly, however, ERK, JNK, and NF-kappaB remain in the cytoplasm in beta4 mutant cells on laminin-5, whereas they enter effectively into the nucleus in the same cells on fibronectin or in wild-type cells on both matrix proteins. Inhibitor studies indicate that alpha6beta4 promotes keratinocyte proliferation and migration through its effect on NF-kappaB and P-JNK. These findings provide evidence that beta4 signaling promotes epidermal growth and wound healing through a previously unrecognized effect on nuclear translocation of NF-kappaB and mitogen-activated protein kinases.     

Seed coats: Structure,development, composition,and biotechnology

Summary Although seeds have been the subject of extensive studies for many years, their seed coats are just beginning to be examined from the perspective of molecular genetics and control of development. The seed coat, plays a vital role in the life cycle of plants by controlling the development of the embryo and determining seed dormancy and germination. Within the seed coat are a number of unique tissues that undergo differentiation to serve specific functions in the seed. A large number of genes are known to be specifically expressed within the seed coat tissues; however, very few of them are understood functionally. The seed coat synthesizes a wide range of novel compounds that may serve the plant in diverse ways, including defense and control of development. Many of the compounds are sources of industrial products and are components of food and feeds. The use of seed coat biotechnology to enhance seed quality and yield, or to generate novel components has not been exploited, largely because of lack of knowledge of the genetic systems that govern seed coat development and composition. In this review, we will examine the recent advances in seed coat, biology from the perspective of structure, composition and molecular genetics. We will consider the diverse avenues that are possible for seed coat biotechnology in the future. This review will focus principally on the seed coats of the Brassicaceae and Fabaceae as they allow us to merge the areas of molecular biology, physiology and structure to gain a perspective on the possibilities for seed coat modifications in the future. The authors have contributed equally and are considered first authors.     

Detection and control of aspartimide formation in the synthesis of cyclic peptides

Extensive two-dimensional NMR analysis was employed to characterize the structural identity of the macrocyclic peptide lactam and the imide analog, a major side reaction product when allyl ester was used to protect the side chain of aspartic acid. A straightforward protocol modification was developed to minimize aspartimide formation during the synthesis of cyclic peptides.     

Wnt-mediated regulation of chondrocyte maturation: modulation by TGF-beta

Wnt proteins are expressed during limb morphogenesis, yet their role and mechanism of action remains unclear during long bone growth. Wnt expression, effects and modulation of signaling events by BMP and transforming growth factor-beta (TGF-beta) were evaluated in chick embryonic chondrocytes. Chondrocyte cell cultures underwent spontaneous maturation with increased expression of colX and this was associated with an increase in the expression of multiple Wnts, including Wnt 4, 5a, 8c, and 9a. Both parathyroid hormone related peptide (PTHrP) and TGF-beta inhibited colX, but had disparate effects on Wnt expression. While TGF-beta strongly inhibited all Wnts, PTHrP did not inhibit either Wnt8c or Wnt9a and had lesser effects on the expression of the other Wnts. BMP-2 induced colX expression, and also markedly increased Wnt8c expression. Overexpression of beta-catenin and/or T cell factor (TCF)-4 also induced the type X collagen promoter. Overexpression of Wnt8c induced maturation, as did overexpression of beta-catenin. The Wnt8c/beta-catenin maturational effects were enhanced by BMP-2 and inhibited by TGF-beta. TGF-beta also inhibited activation of the Topflash reporter by beta-catenin, suggesting a direct inhibitory effect since the Topflash reporter contains only beta-catenin binding sequences. In turn beta-catenin inhibited activation of the p3TP-Luc reporter by TGF-beta, although the effect was partial. Thus, Wnt/beta-catenin signaling is a critical regulator of the rate of chondrocyte differentiation. Moreover, this pathway is modulated by members of the TGF-beta family and demonstrates the highly integrated nature of signals controlling endochondral ossification.     

Frequencies of allelic polymorphism of endothelial NO-synthase gene in patients with acute coronary syndrome in Ukrainian population

Endothelial NO-synthase (eNOS) gene allelic polymorphism in 221 patients with acute coronary syndrome and in 83 practically healthy people was determined. It was shown that interrelations of normal homozygotes, heterozygotes and pathologic homozygotes in T/C promoter polymorphism analysis accout 48%, 36% and 16% correspondingly (in control--48%, 46%, 6%; P < 0.05 by chi2-test); in G894 --> T polymorphism ofexon 7 analysis--34%, 58%, 8% (in control--29%, 67%, 4%; P > 0.05), and in determination of 4a/4b polymorphism of intron 4--64.5%, 31% and 4.5% (in control--62.5%, 32.5%, 5%; P > 0.05). Obtained data show that eNOS C/C promoter variant is a risk factor of acute coronary syndrome in Ukrainian population.     

Virus-induced inhibition of CD1d1-mediated antigen presentation: reciprocal regulation by p38 and ERK

A critical component of the host's innate immune response involves lipid Ag presentation by CD1d molecules to NK T cells. In this study we used murine CD1d1-transfected L (L-CD1) cells to study the effect of viruses on CD1d-mediated Ag presentation to NKT cells and found that an infection with vesicular stomatitis and vaccinia (but not lymphocytic choriomeningitis) virus inhibited murine CD1d1-mediated Ag presentation. This was under the reciprocal control of the MAPKs, p38 and ERK, and was due to changes in the intracellular trafficking of CD1d1. The reciprocal regulation of CD1d1-mediated Ag presentation by MAPK suggests that the targeting of these pathways is a novel means of immune evasion by viruses.     

Estrogen regulates CCR gene expression and function in T lymphocytes

Estrogen has been implicated in the observed female bias in autoimmune diseases. However, the mechanisms behind this gender dimorphism are poorly defined. We have previously reported that in vivo T cell trafficking is gender- and estrogen-dependent. Chemokine receptors are critical determinants of T cell homing and immune response. In this study, we show that the female gender is associated with increased CD4(+) T cell CCR1-CCR5 gene and protein expression in mice. The increased CCR expression correlates with enhanced in vitro chemotaxis response to MIP-1beta (CCL4). In vivo treatment of young oophorectomized and postmenopausal female mice with 17beta-estradiol also increased CD4(+) T cell CCR expression. Finally, 17beta-estradiol enhances tyrosine phosphorylation in T cells stimulated with MIP-1alpha in a time-dependent manner. Our results indicate an important role of estrogen in determining T cell chemokine response that may help explain the increased susceptibility and severity of autoimmune diseases in females.