Microtubules in epidermal and cortical root cells of Brassica rapa during clinorotation

Using confocal microscopy the organization of tubulin cytoskeleton including endoplasmic and cortical microtubules (CMTs) has been studied in epidermal and cortical cells of the different growth zones of main root of Brassica rapa L. 6-days-old seedlings in control conditions and under clinorotation. It was shown that changes in CMTs orientation occured only in the distal elongation zone (DEZ). In the control, CMT arrays oriented transversely to the root long axis. Under clinorotation appearance of the shorter randomly organized CMTs was observed. Simultaneously, a significant decrease in the cell length in the central elongation zone (CEZ) under clinorotation was detected. It is suggested that the decline of anisotropic growth typical for CEZ cells is connected with CMTs disorientation under clinorotation.     

Amino acid residues that determine functional specificity of NADP- and NAD-dependent isocitrate and isopropylmalate dehydrogenases

Isocitrate and isopropylmalalte dehydrogenases are homologous enzymes important for the cell metabolism. They oxidize their substrates using NAD or NADP as cofactors. Thus, they have two specificities, towards the substrate and the cofactor, appearing in three combinations. Although many three-dimensional (3D) structures are resolved, identification of amino acids determining these specificities remains a challenge. We present computational identification and analysis of specificity-determining positions (SDPs). Besides many experimentally proven SDPs, we predict new SDPs, for example, four substrate-specific positions (103Leu, 105Thr, 337Ala, and 341Thr in IDH from E. coli) that contact the cofactor and may play a role in the recognition process.     

Homodimerization Controls the Fibroblast Growth Factor 9 Subfamily's Receptor Binding and Heparan Sulfate-Dependent Diffusion in the Extracellular Matrix

Uncontrolled fibroblast growth factor (FGF) signaling can lead to human diseases, necessitating multiple layers of self-regulatory control mechanisms to keep its activity in check. Herein, we demonstrate that FGF9 and FGF20 ligands undergo a reversible homodimerization, occluding their key receptor binding sites. To test the role of dimerization in ligand autoinhibition, we introduced structure-based mutations into the dimer interfaces of FGF9 and FGF20. The mutations weakened the ability of the ligands to dimerize, effectively increasing the concentrations of monomeric ligands capable of binding and activating their cognate FGF receptor in vitro and in living cells. Interestingly, the monomeric ligands exhibit reduced heparin binding, resulting in their increased radii of heparan sulfate-dependent diffusion and biologic action, as evidenced by the wider dilation area of ex vivo lung cultures in response to implanted mutant FGF9-loaded beads. Hence, our data demonstrate that homodimerization autoregulates FGF9 and FGF20''s receptor binding and concentration gradients in the extracellular matrix. Our study is the first to implicate ligand dimerization as an autoregulatory mechanism for growth factor bioactivity and sets the stage for engineering modified FGF9 subfamily ligands, with desired activity for use in both basic and translational research.Fibroblast growth factor (FGF) signaling plays pleiotropic roles throughout the life spans of mammalian organisms, ranging from germ cell maturation, mesoderm induction, body plan formation, and organogenesis during embryonic development to serum phosphate homeostasis and glucose, bile acid, lipid, and cholesterol metabolism in the adult (3, 23, 27, 28, 57, 60, 62). The diversity of FGF signaling is underscored by virtue of the fact that aberrant FGF signaling leads to a wide array of human diseases, including skeletal and olfactory/reproductive syndromes, phosphate wasting disorders, and cancer (16, 60, 67). Recent data also implicate dysregulated FGF signaling in the etiology of neurodegenerative disorders, such as major depressive disorder and Parkinson''s disease (10, 63, 64).Based on pairwise sequence homology and phylogeny, the 18 bona fide mammalian FGFs (FGF1 to FGF10 and FGF16 to FGF23) are divided into six subfamilies (45). Five FGF subfamilies have high-to-moderate affinity for pericellular heparan sulfate (HS) glycosaminoglycans and thus diffuse locally within tissues to act in a paracrine fashion, whereas the poor affinity of the FGF19 subfamily for HS enables this subfamily to act in an endocrine manner (28, 38). All FGFs share a core homology region of about 120 amino acids, which fold into 12 antiparallel β strands (β1 to β12) that are arranged into three sets of four-stranded β sheets (β-trefoil fold) (39). The globular FGF core domain is flanked by highly divergent N- and C-terminal extensions, which are the principal regions responsible for the different biology of FGFs.FGFs exert their diverse actions by binding and activating FGF receptors (FGFRs) in an HS-dependent fashion (51, 53, 69). There are four distinct mammalian FGFR genes (FGFR1 to FGFR4), each coding for a single-pass transmembrane tyrosine kinase receptor whose ectodomain consists of three immunoglobulin-like domains (D1 to D3) connected by flexible linkers and whose intracellular domain contains the conserved tyrosine kinase domain flanked by the juxtamembrane (JM) and C-terminal regions (38). The 210-amino-acid-long D2-D3 segment of the ectodomain is both necessary and sufficient for ligand binding (20, 51, 52, 58, 70).FGF signaling is tightly regulated by spatial and temporal expression of ligands, receptors, HS cofactors, and most critically by means of FGF-FGFR binding specificity. The tissue-specific alternative splicing in the D3 domain of FGFR1 to FGFR3 is the main mechanism by which FGF-FGFR binding specificity is regulated. This splicing event gives rise to epithelial “b” isoforms (FGFR1b to FGFR3b) and mesenchymal “c” isoforms (FGFR1c to FGFR3c) (24, 25, 47, 68), which differ from one another at the primary sequences of their key ligand binding regions and thus in their FGF binding specificity/promiscuity profiles. Most FGFs are also expressed in either epithelial or mesenchymal tissues and exhibit specificity for FGFR isoforms expressed in the opposite tissues. This results in the establishment of a bidirectional signaling loop between the epithelium and mesenchyme that is essential for organogenesis and tissue homeostasis. It is well established that FGF7 and FGF10, which are expressed exclusively in the mesenchyme, activate specifically FGFR2b to mediate mesenchymal-to-epithelial signaling in the lung, prostate, and lacrimal, mammary, and salivary glands (19, 29, 35, 36, 59). Several lines of genetic and biochemical evidence suggest that the members of the FGF9 subfamily, which includes FGF9, FGF16, and FGF20, convey the reciprocal signaling from the epithelium to the mesenchyme. In the prostate, the epithelial-specific FGF9 has been shown to activate mesenchymal FGFR3c isoforms (25). In the heart, FGF9, FGF16, and FGF20 in the epicardium and endocardium stimulate myocardial proliferation and differentiation in vivo, acting redundantly through FGFR1c and FGFR2c (32). Analysis of FGF9-deficient mice has identified FGF9 as a reciprocal epithelial-to-mesenchymal signal required for morphogenesis of the lung, cecum, small intestine, and inner ear (14, 49, 65, 71). In addition, studies in zebra fish show that FGF16 and FGF20 are apical ectodermal ridge factors that are required for pectoral fin bud outgrowth and, in general, for cell proliferation and differentiation of the mesenchyme (41, 66).In light of the key role of the FGF9 subfamily in tissue homeostasis, it is essential to investigate the molecular mechanisms by which the activity of this subfamily is regulated. Our previous structural and in vitro studies of FGF9 showed that homodimerization masks FGF9''s key receptor binding sites, suggesting that ligand dimerization may autoinhibit FGF9''s biologic activity (50). In this report, we show that, like FGF9, FGF20 also homodimerizes in the crystal and in solution. Characterization of the dimer interface mutations in vitro and in living cells demonstrates that ligand homodimerization autoinhibits FGF9 and FGF20 signaling by suppressing both receptor binding and HS-dependent diffusion in the extracellular matrix (ECM). Our study is the first to implicate ligand dimerization as an autoregulatory mechanism in growth factor bioactivity.     

Sexual cycle properties and changes in carotenoid levels in ovary of Japanese mitten crab <Emphasis Type="Italic">Eriocheir japonica</Emphasis> from rivers of Primorye

The sexual cycle of Japanese mitten crab Eriocheir japonica (de Haan, 1835) in its northern range in water bodies of the Primorye proved to have specific properties including changed seasonal dynamics of gonad maturity stages, delayed spawning period, and reduced breeding season. The ovary color regularly changed during the gonad growth in females, which was accompanied by an increase in total carotenoid content from 1.4 to 22.2 mg/100 g tissue and by changes in the proportion of major pigments. The relative content of the carotene fraction notably decreased and that of astaxanthin slightly decreased during gonad maturation. The revealed changes in carotenoid levels in the ovary point to the activity and significance of pigment metabolism in the reproductive cycle of E. japonica.     

INFLUENCE OF EDUCATION/PROFESSION OF MEXICAN CONSUMERS ON ACCEPTANCE AND PURCHASE INTENT OF CORN TORTILLA

Influence of education/profession of Mexican consumers on acceptance and purchase intent of corn tortillas was evaluated. Two groups of 150 Mexican consumers each (faculty/graduate students [A] versus field laborers [B]) rated sensory acceptability of corn tortillas. Acceptance and purchase intent were rated using a binomial scale. Group B had higher expectations towards corn tortillas as indicated by lower acceptability ratings. Both groups used rollability, resistance-to-tearing, and chewiness to differentiate tortilla samples, but group B paid more attention to these attributes as indicated by higher canonical correlation values. Acceptance was influenced by overall liking for both groups; chewiness and taste were also significant for group B. Purchase intent was influenced by overall liking and taste for both groups, but color for group A, and appearance and chewiness for group B were also critical. This study demonstrated that education/profession of Mexican consumers affected their acceptance and purchase intent of corn tortillas.

PRACTICAL APPLICATIONS

This study revealed that education/profession of Mexican consumers affected acceptance and purchase intent of corn tortillas. Two groups of consumers with different levels of education had different expectation toward tortillas when making purchase decision. Understanding how each consumer segment differently perceives about the tortilla product will give marketers and manufacturers a better direction for developing tortilla products with expected sensory qualities.     

Transgene × Environment Interactions in Genetically Modified Wheat

Background

The introduction of transgenes into plants may cause unintended phenotypic effects which could have an impact on the plant itself and the environment. Little is published in the scientific literature about the interrelation of environmental factors and possible unintended effects in genetically modified (GM) plants.

Methods and Findings

We studied transgenic bread wheat Triticum aestivum lines expressing the wheat Pm3b gene against the fungus powdery mildew Blumeria graminis f.sp. tritici. Four independent offspring pairs, each consisting of a GM line and its corresponding non-GM control line, were grown under different soil nutrient conditions and with and without fungicide treatment in the glasshouse. Furthermore, we performed a field experiment with a similar design to validate our glasshouse results.The transgene increased the resistance to powdery mildew in all environments. However, GM plants reacted sensitive to fungicide spraying in the glasshouse. Without fungicide treatment, in the glasshouse GM lines had increased vegetative biomass and seed number and a twofold yield compared with control lines. In the field these results were reversed. Fertilization generally increased GM/control differences in the glasshouse but not in the field. Two of four GM lines showed up to 56% yield reduction and a 40-fold increase of infection with ergot disease Claviceps purpurea compared with their control lines in the field experiment; one GM line was very similar to its control.

Conclusions

Our results demonstrate that, depending on the insertion event, a particular transgene can have large effects on the entire phenotype of a plant and that these effects can sometimes be reversed when plants are moved from the glasshouse to the field. However, it remains unclear which mechanisms underlie these effects and how they may affect concepts in molecular plant breeding and plant evolutionary ecology.     

Virions and membrane proteins of the potato X virus interact with microtubules and enables tubulin polymerization in vitro

A study was made of the in vitro interactions of virions and the coat protein (CP) of the potato virus X (PVX) with microtubules (MT). Both virions and CP cosedimented with taxol-stabilized MT. In the presence of PVX CP, tubulin polymerized to produce structures resistant to chilling. Electron microscopy revealed the aberrant character of the resulting tubulin polymers (protofilaments and their sheets), which differed from MT assembled in the presence of cell MAP2. In contrast, PVX virions induced the assembly of morphologically normal MT sensitive to chilling. Virions were shown to compete with MAP2 for MT binding, suggesting an overlap for the MT sites interacting with MAP2 and with PVX virions. It was assumed that PVX virions interact with MT in vivo and that, consequently, cytoskeleton elements participate in intracellular compartmentalization of the PVX genome.     

Proliferative activity of neokyotorphin-related hemoglobin fragments in cell cultures

alpha-Hemoglobin fragments alpha-(133-141), alpha-(134-141), alpha-(135-141), alpha-(137-141), alpha-(134-140), alpha-(133-138), alpha-(134-140) and alpha-(137-138) stimulate L929 tumor cell proliferation, alpha-(134-141) being the most active. alpha-(134-141) stimulates proliferation of M3 melanoma cells, murine embryonic fibroblasts, primary cultures of red bone marrow and spleen cells. In L929 cells the effect of alpha-(134-141) is cell density independent; in M3 cells alpha-(137-141) and alpha-(134-141) are most active at density 10,000 cells/well (96 well plate) independently on FBS content.     

Expression, regulation, and pathophysiological role of NAD(P)H oxidases in atherogenesis

Oxidative stress has been implicated in the development and progression of atherosclerotic lesions. Significant increase of reactive oxygen species production by vascular cells can lead to progression of atherosclerotic lesions and development of unstable plaques due to triggering the apoptosis of endothelial and smooth muscle cells, expression of matrix metalloproteases and inflammatory cytokines. Cytolysis NAD(P)H-dependent oxidases appeared to be involved in reactive oxygen species production in the vascular network. Understanding of functions and regulation of individual NAD(P)H oxidases in atherosclerotic lesions can facilitate the development of novel therapeutic strategy for treating atherosclerosis. This review summarizes current data regarding expression, regulation and pathophysiological significance of these enzymes during development and progression of human atherosclerotic lesions.     

Dynamics of antibody nuclease activity in blood of women during pregnancy and lactation

In human milk we previously found catalytic antibodies (abzymes) catalyzing hydrolysis of DNA, RNA, NMP, NDP, and NTP and also phosphorylation of proteins and lipids. In the present study we have analyzed nuclease activities of antibodies in blood of women during pregnancy and lactation. Blood of healthy male and female volunteers lacked catalytically active antibodies, whereas antibodies from blood of pregnant women hydrolyzed DNA and RNA and their relative activity varied over a wide range. Relative blood abzyme activities significantly increased after delivery and at the beginning of lactation. The highest abzyme activity was observed in blood of parturient women. Although the dynamics of changes in antibody DNase activity during pregnancy was rather individual for each woman, there was a common trend in the increase in antibody activity in the first and/or third trimester of the pregnancy. The DNase activity of IgG and IgM from blood of healthy pregnant women was 4-5 times less than that from pregnant women with pronounced autoimmune thyroiditis.