hi2-1, A QTL which improves harvest index, earliness and alters metabolite accumulation of processing tomatoes

Harvest index, defined as the ratio of reproductive yield to total plant biomass, and early ripening are traits with important agronomic value in processing tomatoes. The Solanum pennellii introgression-line (IL) population shows variation for harvest index and earliness. Most of the QTL mapped for these traits display negative agronomic effects; however, hi2-1 is a unique QTL displaying improved harvest index and earliness. This introgression was tested over several years and under different genetic backgrounds. Thirty-one nearly isogenic sub-lines segregating for the 18 cM TG33–TG276 interval were used for fine mapping of this multi-phenotypic QTL. Based on this analysis the phenotypic effects for plant weight, Brix, total yield and earliness were co-mapped to the same region. In a different mapping experiment these sub-lines were tested as heterozygotes in order to map the harvest index QTL which were only expressed in the heterozygous state. These QTL mapped to the same candidate region, suggesting that hi2-1 is either a single gene with pleiotropic effects or represents linked genes independently affecting these traits. Metabolite profiling of the fruit pericarp revealed that a number of metabolic QTL co-segregate with the harvest index trait including those for important transport assimilates such as sugars and amino acids. Analysis of the flowering pattern of these lines revealed induced flowering at IL2-1 plants, suggest that hi2-1 may also affect harvest index and early ripening by changing plant architecture and flowering rate.     

Molecular mechanisms of beta-catenin recognition by adenomatous polyposis coli revealed by the structure of an APC-beta-catenin complex.

The adenomatous polyposis coli (APC) tumor suppressor protein plays a critical role in regulating cellular levels of the oncogene product beta-catenin. APC binds to beta-catenin through a series of homologous 15 and 20 amino acid repeats. We have determined the crystal structure of a 15 amino acid beta-catenin binding repeat from APC bound to the armadillo repeat region of beta-catenin. Although it lacks significant sequence homology, the N-terminal half of the repeat binds in a manner similar to portions of E-cadherin and XTcf3, but the remaining interactions are unique to APC. We discuss the implications of this new structure for the design of therapeutics, and present evidence from structural, biochemical and sequence data, which suggest that the 20 amino acid repeats can adopt two modes of binding to beta-catenin.     

Localized cytosolic alkalization and its functional impact in ciliary cells

Using confocal microscopy we demonstrate that ciliary cells from airway epithelium maintain two qualitatively distinct cytosolic regions in terms of pH regulation. While the bulk of the cytosol is stringently buffered and is virtually insensitive to changes in extracellular pH (pHo), the values of cytosolic pH in the vicinity of the ciliary membrane is largely determined by pHo. Variation of pHo from 6.2 up to 8.5 failed to affect ciliary beat frequency (CBF). Application of NH(4)Cl induced profound localized alkalization near cilia, which did not depress ciliary activity, but resulted in strong and prolonged enhancement of CBF. Calmodulin and protein kinase A (PKA) functionality was essential for the alkalization-induced CBF enhancement. We suggest that the ability of airway epithelium to sustain unusually strong but localized cytosolic alkalization near cilia facilitates CBF enhancement through altering the binding constants of Ca2+ to calmodulin and promotion of Ca2+-calmodulin complex formation. The NH4Cl-induced elevations in cytosolic pH and Ca2+ concentration act synergistically to activate calmodulin-dependent processes, cAMP pathway, and, thereby, stimulate CBF.     

Melting of crosslinked DNA: VI. Comparison of influence of interstrand crosslinks and other chemical modifications formed by antitumor compounds on DNA stability

A computer modeling of thermodynamic properties of a long DNA of N base pairs that includes omega interstrand crosslinks (ICLs), or omega chemical modifications involving one strand (monofunctional adducts, intrastrand crosslinks) has been carried out. It is supposed in our calculation that both types of chemical modifications change the free energy of the helix-coil transition at sites of their location by deltaF. The value deltaF>0 corresponds to stabilization, i.e., to the increase in melting temperature. It is also taken into account that ICLs form additional loops in melted regions and prohibit strand dissociation after full DNA melting. It is shown that the main effect of interstrand crosslinks on the stability of long DNA's is caused by the formation of additional loops in melted regions. This formation increases DNA melting temperature (T(m)) much stronger than replacing omega base pairs of AT type with GC. A prohibition of strand dissociation after crosslinking, which strongly elevates the melting temperature of oligonucleotide duplexes, does not influence melting behavior of long DNA's (N>/=1000 bp). As was demonstrated earlier for the modifications involving one or the other strand, the dependence of the shift of melting temperature deltaT(m) on the relative number of modifications r = omega/(2N) is a linear function for any deltaF, and deltaT(m)(r) identical with 0 for the ideal modifications (deltaF=0). We have shown that deltaT(m)(r) is the same for periodical and random distribution if the absolute value of deltaF is less 2 kcal. The absolute value of deltaT(m)(r) at deltaF>2 kcal and deltaF<-2 kcal is higher for periodical distribution. For interstrand crosslinks, the character of the dependence deltaT(m)(r) is quite different. It is nonlinear, and the shape of the corresponding curve is strongly dependent on deltaF. For "ideal" interstrand crosslinks (deltaF=0), the function deltaT(m)(r) is not zero. It is monotone positive nonlinear, and its slope decreases with r. If r<0.004, then the entropy stabilizing effect of interstrand crosslinking itself exceeds the influence of a distortion of the double helix at sites of their location. The resulting deltaT(m)(r) is positive even in the case of the infinite destabilization at sites of the ICLs (deltaF--> -infinity). In general, stabilizing influence of interstrand crosslinks is almost fully compensated for by local structural distortions caused by them if 0相似文献   

Conversion of an MMP-potent scaffold to an MMP-selective HER-2 sheddase inhibitor via scaffold hybridization and subtle P1' permutations

A series of beta-sulfonamide piperidine hydroxamates were prepared and shown to be potent inhibitors of the human epidermal growth factor receptor-2 (HER-2) sheddase with excellent selectivity against MMP-1, -2, -3, and -9. This was achieved by exploiting subtle differences within the otherwise highly conserved S(1)(') binding pocket of the active sites within the metalloprotease family. In addition, it was discovered that the introduction of polarity to the P(1) and P(1)(') groups reduced the projected human clearance.     

Identification and role of the homodimerization interface of the glycosylphosphatidylinositol-anchored membrane type 6 matrix metalloproteinase (MMP25)

The membrane type (MT) 6 matrix metalloproteinase (MMP) (MMP25) is a glycosylphosphatidylinositol-anchored matrix metalloproteinase (MMP) that is highly expressed in leukocytes and in some cancer tissues. We previously showed that natural MT6-MMP is expressed on the cell surface as a major reduction-sensitive form of M(r) 120, likely representing enzyme homodimers held by disulfide bridges. Among the membrane type-MMPs, the stem region of MT6-MMP contains three cysteine residues at positions 530, 532, and 534 which may contribute to dimerization. A systematic site-directed mutagenesis study of the Cys residues in the stem region shows that Cys(532) is involved in MT6-MMP dimerization by forming an intermolecular disulfide bond. The mutagenesis data also suggest that Cys(530) and Cys(534) form an intramolecular disulfide bond. The experimental observations on cysteines were also investigated by computational studies of the stem peptide, which validate these proposals. Dimerization is not essential for transport of MT6-MMP to the cell surface, partitioning into lipid rafts or cleavage of alpha-1-proteinase inhibitor. However, monomeric forms of MT6-MMP exhibited enhanced autolysis and metalloprotease-dependent degradation. Collectively, these studies establish the stem region of MT6-MMP as the dimerization interface, an event whose outcome imparts protease stability to the protein.     

Role of small loops in DNA melting

Short melted regions less than 100 base pairs (bp) in length are rarely found in the differential melting curves (DMC) of natural DNAs. Therefore, it is supposed that their characteristics do not affect DNA melting behavior. However, in our previous study, a strong influence of the form of the entropy factor of small loops on melting of cross-linked DNAs was established (D. Y. Lando, A. S. Fridman et al., Journal of Biomolecular Structure and Dynamics, 1997, Vol. 15, pp. 141-150; Journal of Biomolecular Structure and Dynamics, 1998, Vol. 16, pp. 59-67). Quite different dependencies of the melting temperature on the relative concentration of interstrand cross-links were obtained for the loop entropy factors given by the Fixman-Freire (Jacobson-Stockmayer) and Wartell-Benight relations. In the present study, the influence of the entropy factor of small loops on the melting of natural DNAs, cross-linked DNAs and periodical double-stranded polynucleotides is compared using computer simulation. A fast combined computational method for calculating DNA melting curves was developed for this investigation. It allows us to assign an arbitrary dependence of the loop entropy factor on the length of melted regions for the terms corresponding to small loops (less than tau bp in length). These terms are calculated using Poland's approach. The Fixman-Freire approach is used for long loops. Our calculations have shown that the temperature dependence of the average length of interior melted regions (loops) has a maximum at T approximately T(m) (T(m) is the DNA melting temperature) in contrast to the dependence of the total average length of melted regions, which increases almost monotonously. Computer modeling demonstrates that prohibition of formation of loops less than tau base pairs in length does not markedly change the DMC for tau < 150 bp. However, the same prohibition strongly affects the average length of internal melted regions for much smaller tau's. The effect is already noticeable for tau = 1 bp and increases with tau. A tenfold increase in the entropy factor of all loops with length less than tau bp causes a noticeable alteration of the DMC for tau > or = 30 bp. It is shown that DMCs are identical for the Wartell-Benight and for the Fixman-Freire (Jacobson-Stockmayer) form of the loop entropy factor. However, for low degree of denaturation, the average length of internal melted regions is 40% lower for the Wartell-Benight form due to the fluctuational opening of short AT-rich regions less than 10 bp in length. The same calculations carried out for periodical polynucleotides demonstrate a much stronger difference in melting behavior for different forms of entropy factors of short loops. The strongest difference occurs if the length of stable GC-rich and unstable AT-rich stretches is equal to 30 bp. However, the comparison carried out in this work demonstrates that the entropy factor of short loops influences melting behavior of cross-linked DNA much stronger than of unmodified DNA with random or periodical sequences.     

Insufficient phosphorylation prevents fc gamma RIIB from recruiting the SH2 domain-containing protein-tyrosine phosphatase SHP-1

Fc gamma RIIB are IgG receptors that inhibit immunoreceptor tyrosine-based activation motif (ITAM)-dependent cell activation. Inhibition depends on an immunoreceptor tyrosine-based inhibition motif (ITIM) that is phosphorylated upon Fc gamma RIIB coaggregation with ITAM-bearing receptors and recruits SH2 domain-containing phosphatases. Agarose bead-coated phosphorylated ITIM peptides (pITIMs) bind in vitro the single-SH2 inositol 5-phosphatases (SHIP1 and SHIP2) and the two-SH2 protein tyrosine phosphatases (SHP-1 and SHP-2). Phosphorylated Fc gamma RIIB, however, recruit selectively SHIP1/2 in vivo. We aimed here at explaining this discordance. We found that beads coated with low amounts of pITIM bound in vitro SHIP1, but not SHP-1, i.e. behaved as phosphorylated Fc gamma RIIB in vivo. The reason is that SHP-1 requires its two SH2 domains to bind on adjacent pITIMs. Consequently, the binding of SHP-1, but not of SHIP1, increased with pITIM density on beads. When trying to increase Fc gamma RIIB phosphorylation in B cells and mast cells, we found that concentrations of ligands optimal for Fc gamma RIIB phosphorylation failed to induce SHP-1 recruitment. SHP-1 was, however, recruited by Fc gamma RIIB when hyperphosphorylated following cell treatment with pervanadate. Our data suggest that Fc gamma RIIB phosphorylation may not be sufficient in vivo to enable the recruitment of SHP-1 but that (pathological?) conditions that would hyperphosphorylate Fc gamma RIIB might enable SHP-1 recruitment.