The regulator of G-protein signaling proteins involved in sugar and abscisic acid signaling in Arabidopsis seed germination

The regulator of G-protein signaling (RGS) proteins, recently identified in Arabidopsis (Arabidopsis thaliana; named as AtRGS1), has a predicted seven-transmembrane structure as well as an RGS box with GTPase-accelerating activity and thus desensitizes the G-protein-mediated signaling. The roles of AtRGS1 proteins in Arabidopsis seed germination and their possible interactions with sugars and abscisic acid (ABA) were investigated in this study. Using seeds that carry a null mutation in the genes encoding RGS protein (AtRGS1) and the alpha-subunit (AtGPA1) of the G protein in Arabidopsis (named rgs1-2 and gpa1-3, respectively), our genetic evidence proved the involvement of the AtRGS1 protein in the modulation of seed germination. In contrast to wild-type Columbia-0 and gpa1-3, stratification was found not to be required and the after-ripening process had no effect on the rgs1-2 seed germination. In addition, rgs1-2 seed germination was insensitive to glucose (Glc) and sucrose. The insensitivities of rgs1-2 to Glc and sucrose were not due to a possible osmotic stress because the germination of rgs1-2 mutant seeds showed the same response as those of gpa1-3 mutants and wild type when treated with the same concentrations of mannitol and sorbitol. The gpa1-3 seed germination was hypersensitive while rgs1-2 was less sensitive to exogenous ABA. The different responses to ABA largely diminished and the inhibitory effects on seed germination by exogenous ABA and Glc were markedly alleviated when endogenous ABA biosynthesis was inhibited. Hypersensitive responses of seed germination to both Glc and ABA were also observed in the overexpressor of AtRGS1. Analysis of the active endogenous ABA levels and the expression of NCED3 and ABA2 genes showed that Glc significantly stimulated the ABA biosynthesis and increased the expression of NCED3 and ABA2 genes in germinating Columbia seeds, but not in rgs1-2 mutant seeds. These data suggest that AtRGS1 proteins are involved in the regulation of seed germination. The hyposensitivity of rgs1-2 mutant seed germination to Glc might be the result of the impairment of ABA biosynthesis during seed germination.     

Cloning of Transglutaminase Gene from Streptomyces fradiae and its Enhanced Expression in the Original Strain

The transglutaminase (TGase) gene of Streptomyces fradiae was cloned. It had an ORF of 1242 bp, encoding a presumed prepro-region of 82 amino acids and a mature TGase of 331 amino acids. Enhanced expression of the TGase was achieved by introducing another copy of TGase gene into the original host genome which was driven by the strong constitutive promoter, “ermE up”, and shown to be expressed at the mRNA and protein levels. TGase activity in the recombinant strain (3.2 U/ml) was improved 1.3-fold when compared to that normally expressed in the original strain (2.4 U/ml). The specific enzyme activity in the recombinant strain (3.8 U/mg) was double that of the original strain (1.9 U/mg).     

Compensatory increase in hepatic lipogenesis in mice with conditional intestine-specific Mttp deficiency

Microsomal TG transfer protein (MTTP) is required for the assembly and secretion of TG (TG)-rich lipoproteins from both enterocytes and hepatocytes. Liver-specific deletion of Mttp produced a dramatic reduction in plasma very low density lipoprotein-TG and virtually eliminated apolipoprotein B100 (apoB100) secretion yet caused only modest reductions in plasma apoB48 and apoB48 secretion from primary hepatocytes. These observations prompted us to examine the phenotype following intestine-specific Mttp deletion because murine, like human enterocytes, secrete virtually exclusively apoB48. We generated mice with conditional Mttp deletion in villus enterocytes (Mttp-IKO), using a tamoxifen-inducible, intestine-specific Cre transgene. Villus enterocytes from chow-fed Mttp-IKO mice contained large cytoplasmic TG droplets and no chylomicron-sized particles within the secretory pathway. Chow-fed, Mttp-IKO mice manifested steatorrhea, growth arrest, and decreased cholesterol absorption, features that collectively recapitulate the phenotype associated with abetalipoproteinemia. Chylomicron secretion was reduced dramatically in vivo, in conjunction with an approximately 80% decrease in apoB48 secretion from primary enterocytes. Additionally, although plasma and hepatic cholesterol and TG content were decreased, Mttp-IKO mice demonstrated a paradoxical increase in both hepatic lipogenesis and very low density lipoprotein secretion. These findings establish distinctive features for MTTP involvement in intestinal chylomicron assembly and secretion and suggest that hepatic lipogenesis undergoes compensatory induction in the face of defective intestinal TG secretion.     

DCC-dependent phospholipase C signaling in netrin-1-induced neurite elongation

Netrins, a family of secreted molecules, play important roles in axon pathfinding during nervous system development. Although phosphatidylinositol signaling has been implicated in this event, how netrin-1 regulates phosphatidylinositol signaling remains poorly understood. Here we provide evidence that netrin-1 stimulates phosphatidylinositol bisphosphate hydrolysis in cortical neurons. This event appears to be mediated by DCC (deleted in colorectal cancer), but not neogenin or Unc5h2. Netrin-1 induces phospholipase Cgamma (PLCgamma) tyrosine phosphorylation. Inhibition of PLC activity attenuates netrin-1-induced cortical neurite outgrowth. These results suggest that netrin-1 regulates phosphatidylinositol turnover and demonstrate a crucial role of PLC signaling in netrin-1-induced neurite elongation.     

The state transition mechanism - simply depending on light-on and -off in Spirulina platensis

The state transition in cyanobacteria is a long-discussed topic of how the photosynthetic machine regulates the excitation energy distribution in balance between the two photosystems. In the current work, whether the state transition is realized by "mobile phycobilisome (PBS)" or "energy spillover" has been clearly answered by monitoring the spectral responses of the intact cells of the cyanobacterium Spirulina platensis. Firstly, light-induced state transition depends completely on a movement of PBSs toward PSI or PSII while the redox-induced one on not only the "mobile PBS" but also an "energy spillover". Secondly, the "energy spillover" is triggered by dissociation of PSI trimers into the monomers which specially occurs under a case from light to dark, while the PSI monomers will re-aggregate into the trimers under a case from dark to light, i.e., the PSI oligomerization is reversibly regulated by light switch on and off. Thirdly, PSI oligomerization is regulated by the local H(+) concentration on the cytosol side of the thylakoid membranes, which in turn is regulated by light switch on and off. Fourthly, PSI oligomerization change is the only mechanism for the "energy spillover". Thus, it can be concluded that the "mobile PBS" is a common rule for light-induced state transition while the "energy spillover" is only a special case when dark condition is involved.     

Purification and some properties of β-N-Acetyl-D-glucosaminidase from viscera of green crab (<Emphasis Type="Italic">Scylla serrata</Emphasis>)

β-N-Acetyl-D-glucosaminidase was purified from viscera of green crab (Scylla serrata) by extraction with 0.01 M Tris-HCl buffer (pH 7.5) containing 0.2 M NaCl, ammonium sulfate fractionation, and then chromatography on Sephadex G-100 and DEAE-cellulose (DE-32). The purified enzyme showed a single band on polyacrylamide gel electrophoresis, and the specific activity was determined to be 7990 U/mg. The molecular weight of the whole enzyme was determined to be 132.0 kD, and the enzyme is composed of two identical subunits with molecular mass of 65.8 kD. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-β-D-glucosaminide (pNP-NAG) were found to be at pH 5.6 and at 50°C, respectively. The study of its stability showed that the enzyme is stable in the pH range from 4.6 to 8.6 and at temperatures below 45°C. The kinetic behavior of the enzyme in the hydrolysis of pNP-NAG followed Michaelis-Menten kinetics with K_m of 0.424 ± 0.012 mM and V_max of 17.65 ± 0.32 µmol/min at pH 5.8 and 37°C, and the activation energy was determined to be 61.32 kJ/mol. The effects of some metal ions on the enzyme were surveyed, and the results show that Na⁺ and K⁺ have no effects on the enzyme activity; Mg²⁺ and Ca²⁺ slightly activate the enzyme, while Ba²⁺, Zn²⁺, Mn²⁺, Hg²⁺, Pb²⁺, Cu²⁺, and Al³⁺ inhibit the enzyme to different extents.     

The role of PDE4 in pulmonary inflammation and goblet cell hyperplasia in allergic rats

Phosphodiesterase 4 (PDE4) has been suggested to a critical factor in the pathogenesis of inflammation by metabolizing cAMP in human leukocytes, endothelium and epithelium. The present study aimed at evaluating the PDE4 activity and expression, the relationship between the inflammation and cAMP- activity in the lungs, and potential interventions of PDE inhibitors and antiinflammatory drugs in the reduction of lung inflammation and goblet cell hyperplasia in allergic rats. The total leukocyte number and eosinophil number in bronchoalveolar lavegar fluid and infiltration of inflammatory cells in the perivascular and peribronchial spaces, structure changes and goblet cell hyperplasia in the OVA-sensitized and challenged allergic rats. A significant correlation was observed between the increases in cAMP-PDE activity and inflammation in the lung. Those OVA-induced changes were prevented by pretreatment with PDE inhibitor in a dose-related patterns and with glucocorticosteriod. We found an increase in the proportion of PDE4 and PDE4 gene expression, while a decrease in the proportion of PDE3 in the lung of the allergic rats. Incubation with different PDE inhibitors down-regulated OVA-induced cAMP hydrolysis. Our data suggest that PDE4C may play an important role in the airway inflammation, remodeling and goblet cell hyperplasia after repeated challenge of sensitized rats.     

Optimization of the cell seeding density and modeling of cell growth and metabolism using the modified Gompertz model for microencapsulated animal cell culture

Cell microencapsulation is one of the promising strategies for the in vitro production of proteins or in vivo delivery of therapeutic products. In order to design and fabricate the optimized microencapsulated cell system, the Gompertz model was applied and modified to describe the growth and metabolism of microencapsulated cell, including substrate consumption and product formation. The Gompertz model successfully described the cell growth kinetics and the modified Gompertz models fitted the substrate consumption and product formation well. It was demonstrated that the optimal initial cell seeding density was about 4-5 x 10(6) cells/mL of microcapsule, in terms of the maximum specific growth rate, the glucose consumption potential and the product formation potential calculated by the Gompertz and modified Gompertz models. Modeling of cell growth and metabolism in microcapsules provides a guideline for optimizing the culture of microencapsulated cells.