Polymorphism in rice amylases at an early stage of seed germination

A polymorphism in rice amylases at an early stage of seed germination is analyzed by zymogram. In non-glutinous cultivars of rice, alpha-amylase isozymes are mainly confirmed in germinating seeds. However, in glutinous cultivars, beta-amylase isozymes, which are not confirmed in nonglutinous cultivars, make up the major part of the total amylase activity and the expression of alpha-amylases are repressed.     

Cytoplasmic surface structure of bacteriorhodopsin consisting of interhelical loops and C-terminal alpha helix, modified by a variety of environmental factors as studied by (13)C-NMR.

We have examined the (13)C-NMR spectra of [3-(13)C] Ala-labeled bacteriorhodopsin and its mutants by varying a variety of environmental or intrinsic factors such as ionic strength, temperature, pH, truncation of the C-terminal alpha helix, and site-directed mutation at cytoplasmic loops, in order to gain insight into a plausible surface structure arising from the C-terminal alpha helix and loops. It is found that the surface structure can be characterized as a complex stabilized by salt bridges or metal-mediated linkages among charged side chains. The surface complex in bacteriorhodopsin is most pronounced under the conditions of 10 mM NaCl at neutral pH but is destabilized to yield relaxed states when environmental factors are changed to high ionic strength, low pH and higher temperature. These two states were readily distinguished by associated spectral changes, including suppressed (cross polarization-magic angle spinning NMR) or displaced (upfield) (13)C signals from the C-terminal alpha helix, or modified spectral features in the loop region. It is also noteworthy that such spectral changes, when going from the complexed to relaxed states, occur either when the C-terminal alpha helix is deleted or site-directed mutations were introduced at a cytoplasmic loop. These observations clearly emphasize that organization of the cytoplasmic surface complex is important in the stabilization of the three-dimensional structure at ambient temperature, and subsequently plays an essential role in biological functions.     

Repression of porin synthesis by salicylate in Escherichia coli, Klebsiella pneumoniae and Serratia marcescens

The synthesis of the OmpF porin in Escherichia coli K-12 was highly and reversibly inhibited by 5 mM salicylate in the bacterial growth medium, and salicylate also inhibited the OmpC porin synthesis, although only weakly. The full expression of the salicylate effect was presumed to require the ompB gene product on comparison between the wild type and ompB mutant strains. The salicylate effect was also observed for the porin protein synthesis in Klebsiella pneumoniae and Serratia marcescens, although an ompB-like gene remains to be identified in both species.     

Secretory and continuous expression of Aspergillus niger glucose oxidase gene in Pichia pastoris

We proposed a yeast transformant cell incorporating the Aspergillus niger glucose oxidase gene (GOX gene), which is capable of constitutively as well as secretory expression. The GOX gene has been cloned in this study. This conclusion is based on the following: first, the ligated DNA determined by electrophoresis, was a 1489-1882bp fragment, close to the size of glucose oxidase (GOD), which is 1818bp. Secondly, the single open reading frame encoded a protein of 605 amino acids. Thirdly, secreted GOD recombinant proteins in the culture supernatants of the GOX gene transformant migrated as a single band in SDS-PAGE with an apparent molecular mass of between 75,000 and 100,000 Da, which is glycosylated GOD by the Pichia pastoris X-33 host machinery during the secretion process. Finally, the clones were cultured and secreted a protein, which possessed the GOD activity of catalyzing beta-d-glucose oxidation. With regard to the pH characteristics, the activity was more than 80% of the maximum activity in the range between pH 5 and pH 7. As for the temperature characteristics, the activity was not less than 92% of the maximum in the temperature range between 10 and 45 degrees C. The GOX gene transformant was able to maintain the GOD enzyme activity and produce recombinant GOD continuously for at least 2 weeks.     

A Dual Role for Corneal Dendritic Cells in Herpes Simplex Keratitis: Local Suppression of Corneal Damage and Promotion of Systemic Viral Dissemination

The cornea is the shield to the foreign world and thus, a primary site for peripheral infections. However, transparency and vision are incompatible with inflammation and scarring that may result from infections. Thus, the cornea is required to perform a delicate balance between fighting infections and preserving vision. To date, little is known about the specific role of antigen-presenting cells in viral keratitis. In this study, utilizing an established murine model of primary acute herpes simplex virus (HSV)-1 keratitis, we demonstrate that primary HSV keratitis results in increased conventional dendritic cells (cDCs) and macrophages within 24 hours after infection. Local depletion of cDCs in CD11c-DTR mice by subconjuntival diphtheria toxin injections, led to increased viral proliferation, and influx of inflammatory cells, resulting in increased scarring and clinical keratitis. In addition, while HSV infection resulted in significant corneal nerve destruction, local depletion of cDCs resulted in a much more severe loss of corneal nerves. Further, local cDC depletion resulted in decreased corneal nerve infection, and subsequently decreased and delayed systemic viral transmission in the trigeminal ganglion and draining lymph node, resulting in decreased mortality of mice. In contrast, sham depletion or depletion of macrophages through local injection of clodronate liposomes had neither a significant impact on the cornea, nor an effect on systemic viral transmission. In conclusion, we demonstrate that corneal cDCs may play a primary role in local corneal defense during viral keratitis and preserve vision, at the cost of inducing systemic viral dissemination, leading to increased mortality.