Rapid evolution of T2/S-RNase genes in Fragaria linked to multiple transitions from self-incompatibility to self-compatibilityOA

The T2/RNase gene family is widespread in eukaryotes,and particular members of this family play critical roles in the gametophytic self-incompatibility(GSI) system in plants.Wild diploid strawberry(Fragaria)species have diversified their sexual systems via self-incompatible and self-compatible traits,yet how these traits evolved in Fragaria remains elusive.By integrating the published and de novo assembled genomes and the newly generated RNA-seq data,members of the RNase T2 gene family were syst...     

Involvement of the MAP kinase pathways in induction of GADD45 following UV radiation

Indian hedgehog (Ihh) is highly expressed in prehypertrophic chondrocytes in vivo and has been proposed to regulate the proliferation and maturation of chondrocytes and bone collar formation in the growth plate. In high-density cultures of rabbit growth-plate chondrocytes, Ihh mRNA was also expressed at the highest level in the prehypertrophic stage. To explore endogenous factors that regulate Ihh expression in chondrocytes, we examined the effects of various growth factors on Ihh mRNA expression in this system. Retinoic acid (RA) and bone morphogenetic protein-2 enhanced Ihh mRNA expression, whereas PTH/PTH-related peptide (PTHrP) markedly suppressed Ihh expression. RA at more than 10(-8) M induced the expression of Ihh and Patched 1 (Ptc1) within 3 h, before it increased the type X collagen mRNA level at 6-24 h. Cycloheximide blocked the up-regulation of Ihh by RA, indicating the requirement of de novo protein synthesis for this stimulation. These findings suggest that RA is involved in the up-regulation of Ihh during endochondral bone formation. In contrast to RA, PTH (1-84) at 10(-7) M abolished the mRNA expression of Ihh and Ptc1 within 2-4 h, before it suppressed the expression of type X collagen at 12-24 h. The inhibition of Ihh expression by PTH (1-84) did not require de novo protein synthesis. PTH (1-34), PTHrP (1-34), and (Bu)(2)cAMP also suppressed Ihh expression. On the other hand, Ihh has been reported to induce PTHrP synthesis in the perichondrium. Consequently, the direct inhibitory action of PTH/PTHrP on Ihh appears to be a negative feedback mechanism that prevents excess PTHrP accumulation in cartilage.     

Comprehensive chemical profiling of gramineous plant root exudates using high-resolution NMR and MS

Root exudates released into soil have important functions in mobilizing metal micronutrients and for causing selective enrichment of plant beneficial soil micro-organisms that colonize the rhizosphere. Analysis of plant root exudates typically has involved chromatographic methods that rely on a priori knowledge of which compounds might be present. In the research reported here, the combination of multinuclear and 2-D NMR with GC-MS and high-resolution MS provided de novo identification of a number of components directly in crude root exudates of different plant types. This approach was applied to examine the role of exudate metal ion ligands (MIL) in the acquisition of Cd and transition metals by barley and wheat. The exudation of mugineic acids and malate was enhanced by Fe deficiency. which in turn led to an increase in the tissue content of Cu, Mn, and Zn. The presence of elevated Cd maintained at a free activity pCd of 8.8 (10(-8.8) M), resulted in reduced phytosiderophore production by Fe deficient plants. The buffer morpholinoethane sulfonate (MES), which is commonly used in chelator-buffering nutrient solutions, was detected in the root exudate mixture, suggesting uptake and re-secretion of this compound by the roots. The ability to detect this compound in complex mixtures containing organic acids, amino acids, and other substances suggests that the analytical methods used here provide an unbiased method for simultaneous detection of all major components contained in root exudates.     

Fast repair of hydroxy radical purine deoxynucleotide adducts by phenylpropanoid glycosides and their derivatives from Chinese herbs

DNA damaged by oxygen radicals has been implicated as a causative event in a number of degenerative diseases, including cancer and aging. So it is very significant to look for ways in which either oxygen radicals are scavenged prior to DNA damage or damaged DNA is repaired to supplement the cells' inadequate repair capacity. The repair activities and reaction mechanism of phenylpropanoid glycosides (PPGs) and their derivatives, isolated from Chinese folk medicinal herbs, towards both dGMP-OH* adducts and dAMP-OH* adducts were studied with the pulse radiolytic technique. On pulse irradiation of nitrous oxide saturated 2 mM dGMP or dAMP aqueous solution containing one of the PPGs or their derivatives, the transient absorption spectra of the hydroxyl adduct of dGMP or dAMP decayed with the formation of that of phenoxyl radicals of PPGs or their derivatives within several decades of microseconds after electron pulse irradiation. The result indicated that dGMP or dAMP hydroxyl adducts can be repaired by PPGs or their derivatives. The rate constants of the repair reactions were deduced to be 0.641-1.28 x 10(9) M(-1) s(-1) for dGMP-OH* and 0.2-0.491 x 10(9) M(-1) s(-1) for dAMP-OH*, which positively correlated to the number of phenolic hydroxyl groups in the glycoside structure. A deeper understanding of this new repair mechanism may help researchers to design strategies to prevent and/or intervene more effectively in free radical related diseases.     

Methodological aspects of measuring amino acid uptake in studies with porcine jejunal brush border membrane vesicles

With L-glutamine, as a representative amino acid this study was undertaken to examine the effects of substrate concentrations on initial and equilibrium amino acid uptake and intravesicular volume determined with porcine jejunal brush border membrane vesicles prepared by Mg2+-aggregation and differential centrifugation. Transport measurements (24 degrees C) were conducted by the rapid filtration manual procedure. Glutamine uptake was shown to occur into an osmotically-active space ranging between 1.09-1.58 microl/mg protein with little non-specific membrane binding. At different concentrations (in parentheses), the duration of initial glutamine uptake in both Na+ gradient and Na+-free conditions was 10 s (0.01 mM), 15 s (0.17 mM), and 20 s (1.9 and 9.4 mM), respectively. Substrate concentrations affected the duration of initial uptake, with lower substrate concentrations giving shorter duration for initial amino acid uptake. At different substrate concentrations (in parentheses), the time required to reach equilibrium glutamine uptake was 5 min (0.01 mM), 10 min (0.17 mM), and 60 min (1.9 and 9.4 mM), respectively. Thus, substrate concentrations also affected the time required to reach equilibrium uptake. The higher the substrate concentration, the longer the incubation time needed to reach equilibrium amino acid uptake. At the glutamine concentrations of 0.01, 0.17, 1.9, and 9.4 mM, the average intravesicular volume was estimated to be 1.58+/-0.21, 1.09+/-0.28, 1.24+/-0.18, and 1.36+/-0.21 microl/mg protein, respectively. Substrate concentrations had no effect (p>0.05) on the intravesicular volume of membrane vesicles. In conclusion, in the experiments on amino acid transport kinetics measured with the rapid filtration manual procedure, the incubation time used for measuring the initial uptake rate should be determined from the time course experiments conducted at the lowest substrate concentration used, whereas the intravesicular volume can be obtained from equilibrium uptake measured at any substrate concentrations.     

Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons

A video-rate (30 frames/s) scanning two-photon excitation microscope has been successfully tested. The microscope, based on a Nikon RCM 8000, incorporates a femtosecond pulsed laser with wavelength tunable from 690 to 1050 nm, prechirper optics for laser pulse-width compression, resonant galvanometer for video-rate point scanning, and a pair of nonconfocal detectors for fast emission ratioing. An increase in fluorescent emission of 1.75-fold is consistently obtained with the use of the prechirper optics. The nonconfocal detectors provide another 2.25-fold increase in detection efficiency. Ratio imaging and optical sectioning can therefore be performed more efficiently without confocal optics. Faster frame rates, at 60, 120, and 240 frames/s, can be achieved with proportionally reduced scan lines per frame. Useful two-photon images can be acquired at video rate with a laser power as low as 2.7 mW at specimen with the genetically modified green fluorescent proteins. Preliminary results obtained using this system confirm that the yellow "cameleons" exhibit similar optical properties as under one-photon excitation conditions. Dynamic two-photon images of cardiac myocytes and ratio images of yellow cameleon-2.1, -3.1, and -3.1nu are also presented.     

Using a galactose library for exploration of a novel hydrophobic pocket in the receptor binding site of the Escherichia coli heat-labile enterotoxin

The binding of the B subunits of Escherichia coli heat-labile enterotoxin (LT) to epithelial cells lining the intestines is a critical step for the toxin to invade the host. This mechanism suggests that molecules which possess high affinity to the receptor binding site of the toxin would be good leads for the development of therapeutics against LT. The natural receptor for LT is the complex ganglioside GM1, which has galactose as its terminal sugar. A chemical library targeting a novel hydrophobic pocket in the receptor binding site of LT was constructed based on galactose derivatives and screened for high affinity to the receptor binding site of LT. This screening identified compounds that have 2-3 orders of magnitude higher affinity toward the receptor binding site of LT than the parent compound, galactose. The present findings will pave the way for developing simple and easily synthesizable molecules, instead of complex oligosaccharides, as drugs and/or prophylactics against LT-caused disease.     

BALB/c mice produce blister-causing antibodies upon immunization with a recombinant human desmoglein 3

Pemphigus vulgaris (PV) is an Ab-mediated autoimmune blistering disease of mucotaneous surfaces. Over 95% of the patients with PV express DR4 or DRw6, and the disease is characterized by the presence of autoantibodies directed against desmoglein 3 (Dsg 3), a protein expressed on keratinocytes. An appropriate animal model is required to understand immunoregulation and to address the role of immunogenetic components in the production of pathogenic Abs that are characteristic of PV. Therefore, we turned to the development of a mouse model. Four strains of female mice (BALB/c, DBA/1, SJL/J, and HRS/J) were screened for their ability to produce pathogenic anti-Dsg 3 Abs. We demonstrated that only BALB/c mice immunized with a full-length Dsg 3 can produce pathogenic Abs capable of causing acantholysis of human foreskin in culture and blistering in neonatal mice. This observation suggested that either H-2d or the BALB background contains the immunogenetic makeup necessary for the production of pathogenic anti-Dsg 3 Abs. No correlation was noted between a given isotype and the pathogenic potential of autoantibodies from different strains of mice. Similarly, the pattern of reactivity of Abs with a panel of 46 synthetic peptides that span the entire Dsg 3 failed to reveal any association between binding specificity and the pathogenic potential, and suggested that pathogenic Abs might recognize conformational epitopes. Moreover, our studies showed that the epitopes recognized by pathogenic Abs are contained within the extracellular Dsg 3.     

Secondary structure and stability of the selenocysteine insertion sequences (SECIS) for human thioredoxin reductase and glutathione peroxidase

We have used high resolution NMR and thermodynamics to characterize the secondary structure and stability of the selenocysteine insertion sequences (SECIS) of human glutathione peroxidase (58 nt) and thioredoxin reductase (51 nt). These sequences are members of the two classes of SECIS recently identified with two distinct structures capable of directing selenocysteine incorporation into proteins in eukaryotes. UV melting experiments showed a single cooperative and reversible transition for each RNA, which indicates the presence of stable secondary structures. Despite their large size, the RNAs gave well resolved NMR spectra for the exchangeable protons. Using NOESY, the imino protons as well as the cytosine amino protons of all of the Watson-Crick base pairs were assigned. In addition, a number of non-canonical base pairs including the wobble G.U pairs were identified. The interbase-pair NOEs allowed definition of the hydrogen-bonded structure of the oligonucleotides, providing an experimental model of the secondary structure of these elements. The derived secondary structures are consistent with several features of the predicted models, but with some important differences, especially regarding the conserved sequence motifs.