Genetic and biochemical evidence for involvement of HOTHEAD in the biosynthesis of long-chain α-,ω-dicarboxylic fatty acids and formation of extracellular matrix

In plants, extracellular matrix polymers built from polysaccharides and cuticular lipids have structural and protective functions. The cuticle is found to be ten times thinner in Arabidopsis thaliana (L.) Heynh than in many other plants, and there is evidence that it is unusual in having a high content of α-,ω-dicarboxylic fatty acids (FAs) in its polyesters. We designated the new organ fusion mutant hth-12 after it appeared to be allelic to adhesion of calyx edges (ace) and hothead (hth), upon molecular cloning of the gene by transposon tagging. This mutant is deficient in its ability to oxidize long-chain ω-hydroxy FAs to ω-oxo FAs, which results in leaf polyesters in decreased α-,ω-dicarboxylic FAs and increased ω-hydroxy FAs. These chemical phenotypes lead to disorder of the cuticle membrane structure in hth-12. ACE/HTH is a single-domain protein showing sequence similarity to long-chain FA ω-alcohol dehydrogenases from Candida species, and we hypothesize that it may catalyze the next step after cytochrome P450 FA ω-hydroxylases in the ω-oxidation pathway. We show that ACE/HTH is specifically expressed in epidermal cells. It appears very likely therefore that the changes in the amount of α-,ω-dicarboxylic FAs in hth-12 reflect the different composition of cuticular polyesters. The ACE/HTH gene is also expressed in root epidermal cells which do not form a polyester membrane on the exterior surface, thereby making it possible that the end products of the pathway, α-,ω-dicarboxylic FAs, are generally required for the cross-linking that ensures the integrity of the outer epidermal cell wall.     

Desiccation responses and survival of Sinorhizobium meliloti USDA 1021 in relation to growth phase, temperature, chloride and sulfate availability

AIMS: To identify physical and physiological conditions that affect the survival of Sinorhizobium meliloti USDA 1021 during desiccation. METHODS AND RESULTS: An assay was developed to study desiccation response of S. meliloti USDA 1021 over a range of environmental conditions. We determined the survival during desiccation in relation to (i) matrices and media, (ii) growth phase, (iii) temperature, and (iv) chloride and sulfate availability. CONCLUSIONS: This study indicates that survival of S. meliloti USDA 1021 during desiccation is enhanced: (i) when cells were dried in the stationary phase, (ii) with increasing drying temperature at an optimum of 37 degrees C, and (iii) during an increase of chloride and sulfate, but not sodium or potassium availability. In addition, we resolved that the best matrix to test survival was nitrocellulose filters. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of physical and physiological factors that determine the survival during desiccation of S. meliloti USDA 1021 may aid in (i) the strategic development of improved seed inocula, (ii) the isolation, and (iii) the development of rhizobial strains with improved ability to survive desiccation. Furthermore, this work may provide insights into the survival of rhizobia under drought conditions.     

Improvement of pristinamycin production by genome shuffling and medium optimization for Streptomyces pristinaespiralis

To isolate an improved pristinamycin producing strain of Streptomyces pristinaespiralis, the technique of Genome shuffling was used which resulted in a high-yield recombinant G 3-56 strain. Strain G 3-56 yielded 322 ± 17 mg/L of pristinamycin which was 11.4-fold higher than that of the initial strain and 3.7-fold higher than strain UN-78 which previously had the highest yield of pristinamycin. The genetic characteristics of the recombinant G 3-56 strain was stable as revealed by our subculture experiments. The optimal production medium was determined using the orthogonal matrix method. Under the optimal medium conditions, the maximum yield of pristinamycin was 412 mg/L with about 1.24-fold higher than the original medium.     

Expression of Discoidin Domain Receptor 2 (DDR2) Extracellular Domain in Pichia Pastoris and Functional Analysis in Synovial Fibroblasts and NIT3T3 Cells

Discoidin domain receptor 2 (DDR2) is a kind of protein tyrosine kinases associated with cell proliferation and tumor metastasis, and collagen, identified as a ligand for DDR2, up-regulates matrix metallloproteinase 1 (MMP-1) and MMP-2 expression in cellular matrix. To investigate the roles of DDR2 in destruction of cartilage in rheumatoid arthritis (RA) and tumor metastasis, we tried to express extracellular domain of DDR2 fused with a His tag to increase protein solubility and facilitate purification (without signal peptide and transmembrane domain, designated DR) in Pichia pastoris, purify the expressed protein, and characterize its function, for purpose of future application as a specific DDR2 antagonist. Two clones of relative high expression of His-DR were obtained, After purification by a Ni-NTA (nitric-tri-acetic acid) chromatographic column, soluble fused His-DR over 90% purity were obtained. Competitive binding inhibition assay demonstrated that expressed His-DR could block the binding of DDR2 and natural DDR2 receptors on NIT3T3 and synovial cell surfaces. Results of RT-PCR, Western blotting, and gelatinase zymography showed that His-DR was capable of inhibiting MMP-1 and MMP-2 secretion from NIT3T3 cells and RA synoviocytes stimulated by collagen II. For MMP-1, the inhibitory effect was displayed at the levels of mRNA and protein, whereas for MMP-2 it was demonstrated at the level of protein physiological activity. All these findings suggested that the fused expressed His-DR inhibited the activity of natural DDR2, and relevant MMP-1 and MMP-2 expression in synoviocytes and NIH3T3 cells provoked by collagen II. Wei Zhang and Tianbing Ding equally contributed to this work.     

Protein Interaction between Ameloblastin and Proteasome Subunit α Type 3 Can Facilitate Redistribution of Ameloblastin Domains within Forming Enamel

Enamel is a bioceramic tissue composed of thousands of hydroxyapatite crystallites aligned in parallel within boundaries fabricated by a single ameloblast cell. Enamel is the hardest tissue in the vertebrate body; however, it starts development as a self-organizing assembly of matrix proteins that control crystallite habit. Here, we examine ameloblastin, a protein that is initially distributed uniformly across the cell boundary but redistributes to the lateral margins of the extracellular matrix following secretion thus producing cell-defined boundaries within the matrix and the mineral phase. The yeast two-hybrid assay identified that proteasome subunit α type 3 (Psma3) interacts with ameloblastin. Confocal microscopy confirmed Psma3 co-distribution with ameloblastin at the ameloblast secretory end piece. Co-immunoprecipitation assay of mouse ameloblast cell lysates with either ameloblastin or Psma3 antibody identified each reciprocal protein partner. Protein engineering demonstrated that only the ameloblastin C terminus interacts with Psma3. We show that 20S proteasome digestion of ameloblastin in vitro generates an N-terminal cleavage fragment consistent with the in vivo pattern of ameloblastin distribution. These findings suggest a novel pathway participating in control of protein distribution within the extracellular space that serves to regulate the protein-mineral interactions essential to biomineralization.     

The biennial life strategy in a random environment

In a previous paper (J. Math. Biol. 26, 199–215 (1988)) we calculated the mean and variance of the long-run geometric growth rate of a discrete-time population model with two age classes in a random environment. The formula which was used in that paper as the starting point for the computation of the variance represents only the contribution of the one-period variances. Here we supplement these results by a calculation of the exact variance. All qualitative conclusions reached before are unaffected.     

Structural signatures of enzyme binding pockets from order-independent surface alignment: a study of metalloendopeptidase and NAD binding proteins

Detecting similarities between local binding surfaces can facilitate identification of enzyme binding sites and prediction of enzyme functions, and aid in our understanding of enzyme mechanisms. Constructing a template of local surface characteristics for a specific enzyme function or binding activity is a challenging task, as the size and shape of the binding surfaces of a biochemical function often vary. Here we introduce the concept of signature binding pockets, which captures information on preserved and varied atomic positions at multiresolution levels. For proteins with complex enzyme binding and activity, multiple signatures arise naturally in our model, forming a signature basis set that characterizes this class of proteins. Both signatures and signature basis sets can be automatically constructed by a method called SOLAR (Signature Of Local Active Regions). This method is based on a sequence-order-independent alignment of computed binding surface pockets. SOLAR also provides a structure-based multiple sequence fragment alignment to facilitate the interpretation of computed signatures. By studying a family of evolutionarily related proteins, we show that for metzincin metalloendopeptidase, which has a broad spectrum of substrate binding, signature and basis set pockets can be used to discriminate metzincins from other enzymes, to predict the subclass of metzincins functions, and to identify specific binding surfaces. Studying unrelated proteins that have evolved to bind to the same NAD cofactor, we constructed signatures of NAD binding pockets and used them to predict NAD binding proteins and to locate NAD binding pockets. By measuring preservation ratio and location variation, our method can identify residues and atoms that are important for binding affinity and specificity. In both cases, we show that signatures and signature basis set reveal significant biological insight.     

Matrix attachment regulates Fas-induced apoptosis in endothelial cells: a role for c-flip and implications for anoikis

Survival of endothelial cells is critical for cellular processes such as angiogenesis. Cell attachment to extracellular matrix inhibits apoptosis in endothelial cells both in vitro and in vivo, but the molecular mechanisms underlying matrix-induced survival signals or detachment-induced apoptotic signals are unknown. We demonstrate here that matrix attachment is an efficient regulator of Fas-mediated apoptosis in endothelial cells. Thus, matrix attachment protects cells from Fas-induced apoptosis, whereas matrix detachment results in susceptibility to Fas-mediated cell death. Matrix attachment modulates Fas-mediated apoptosis at two different levels: by regulating the expression level of Fas, and by regulating the expression level of c-Flip, an endogenous antagonist of caspase-8. The extracellular signal-regulated kinase (Erk) cascade functions as a survival pathway in adherent cells by regulating c-Flip expression. We further show that detachment-induced cell death, or anoikis, itself results from activation of the Fas pathway by its ligand, Fas-L. Fas-L/Fas interaction, Fas-FADD complex formation, and caspase-8 activation precede the bulk of anoikis in endothelial cells, and inhibition of any of these events blocks anoikis. These studies identify matrix attachment as a survival factor against death receptor-mediated apoptosis and provide a molecular mechanism for anoikis and previously observed Fas resistance in endothelial cells.