Diversity of Novel Glutenin Subunits in Bread Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Glutenin is a major determinant of baking performance and viscoelasticity, which are responsible for high-quality bread with a light porous crumb structure of a well-leavened loaf. We analyzed the diversity of glutenin genes from six wheat cultivars (Korean cvs. Keumgang and Jinpum, Chinese cvs. China-108 and Yeonnon-78, and Japanese cvs. Norin-61 and Kantou-107). Glutenins contain two types of isoforms such as high molecular weight glutenin subunit (HMW-GS) and low molecular weight glutenin subunit (LMW-GS). Glutenin fractions were extracted from wheat endosperm using Osborne solubility method. A total of 217 protein spots were separated on two-dimensional gel electrophoresis with isoelectric focusing (wide range of pH 3–10). The proteins spots were subjected to tryptic digestion and identified by matrix assisted laser desorption/ionization–time of flight mass spectrometry. HMW-GS (43 isoforms) and LMW-GS (seven isoforms) are directly responsible for producing high-quality bread and noodles. Likewise, all the seed storage proteins are digested to provide nutrients for the embryo during seed germination and seedling growth. We identified the diverse glutenin subunits in wheat cultivars and compared the gluten isoforms among different wheat cultivars according to quality. This work gives an insight on the quality improvement in wheat crop.     

Expression, purification, and characterization of gp160e, the soluble, trimeric ectodomain of the simian immunodeficiency virus envelope glycoprotein, gp160

The envelope glycoprotein, gp160, of simian immunodeficiency virus (SIV) shares approximately 25% sequence identity with gp160 from the human immunodeficiency virus, type I, indicating a close structural similarity. As a result of binding to cell surface CD4 and co-receptor (e.g. CCR5 and CXCR4), both SIV and human immunodeficiency virus gp160 mediate viral entry by membrane fusion. We report here the characterization of gp160e, the soluble ectodomain of SIV gp160. The ectodomain has been expressed in both insect cells and Chinese hamster ovary (CHO)-Lec3.2.8.1 cells, deficient in enzymes necessary for synthesizing complex oligosaccharides. Both the primary and a secondary proteolytic cleavage sites between the gp120 and gp41 subunits of gp160 were mutated to prevent cleavage and shedding of gp120. The purified, soluble glycoprotein is shown to be trimeric by chemical cross-linking, gel filtration chromatography, and analytical ultracentrifugation. It forms soluble, tight complexes with soluble CD4 and a number of Fab fragments from neutralizing monoclonal antibodies. Soluble complexes were also produced of enzymatically deglycosylated gp160e and of gp160e variants with deletions in the variable segments.     

Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Nucleoside diphosphate kinase (NDPK, Nm23) has been implicated as a multifunctional protein. However, the regulatory mechanism of NDPK is poorly understood. We have examined the modification of NDPK in oxidative stresses. We found that oxidative stresses including diamide and H(2)O(2) treatment cause disulfide cross-linking of NDPK inside cells. This cross-linking was reversible in response to mild oxidative stress, and irreversible to strong stress. This suggests that disulfide cross-linked NDPK may be a possible mechanism in the modification of cellular regulation. To confirm this idea, oxidative modification of NDPK has been performed in vitro using purified human NDPK H(2)O(2) inactivated the nucleoside diphosphate (NDP) kinase activity of NDPK by producing intermolecular disulfide bonds. Disulfide cross-linking of NDPK also dissociated the native hexameric structure into a dimeric form. The oxidation sites were identified by the analysis of tryptic peptides of oxidized NDPK, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Intermolecular cross-linking between Cys109-Cys109, which is highly possible based on the X-ray crystal structure of NDPK-A, and oxidations of four methionine residues were identified in H(2)O(2)-treated NDPK. This cross-linkng was confirmed using mutant C109A (NDPK-A(C109A)) which had similar enzymatic activity as a wild NDPK-A. Mutant NDPK-A(C109A) was not cross-linked and was not easily denatured by the oxidant. Therefore, enzymatic activity and the quaternary structure of NDPK appear to be regulated by cross-linking with oxidant. These findings suggest one of the regulatory mechanisms of NDPK in various cellular processes.     

Denitrification is a common feature among members of the genus Bacillus

Although several Gram-positive denitrifiers have been characterized in the past, there is still uncertainty about the occurrence of the denitrification trait among these bacteria. In an isolation campaign on luvisol soil, Bacillus spp. were among the most abundant retrieved cultured denitrifiers next to members of Rhizobiaceae family and genus Cupriavidus. Subsequent screening of 180 representatives of the genus Bacillus (encompassing more than half of the current validly described species diversity in Bacillus) was performed and demonstrated the potential for dissimilatory reduction of nitrogen compounds in 45 of the 87 investigated species, with 19 species containing denitrifying members. The influence of several electron donors and acceptors was tested. The use of more than one electron acceptor, e.g. both nitrate and nitrite, was crucial to detect the denitrification potential of reference strains. Complex electron donors, most suitable for aerobic growth, were ideal for denitrification testing, while retrieval of denitrifiers from the environment was facilitated by the use of defined electron donors, due to less interference of other anaerobic growers. The outcome of the isolation campaign and screening of reference strain set suggest that bacilli may be potential contributors to denitrification in terrestrial and possibly other ecosystems.     

HIV-2 Integrase Variation in Integrase Inhibitor-Na?ve Adults in Senegal,West Africa

Background

Antiretroviral therapy for HIV-2 infection is hampered by intrinsic resistance to many of the drugs used to treat HIV-1. Limited studies suggest that the integrase inhibitors (INIs) raltegravir and elvitegravir have potent activity against HIV-2 in culture and in infected patients. There is a paucity of data on genotypic variation in HIV-2 integrase that might confer intrinsic or transmitted INI resistance.

Methods

We PCR amplified and analyzed 122 HIV-2 integrase consensus sequences from 39 HIV-2–infected, INI-naive adults in Senegal, West Africa. We assessed genetic variation and canonical mutations known to confer INI-resistance in HIV-1.

Results

No amino acid-altering mutations were detected at sites known to be pivotal for INI resistance in HIV-1 (integrase positions 143, 148 and 155). Polymorphisms at several other HIV-1 INI resistance-associated sites were detected at positions 72, 95, 125, 154, 165, 201, 203, and 263 of the HIV-2 integrase protein.

Conclusion

Emerging genotypic and phenotypic data suggest that HIV-2 is susceptible to the new class of HIV integrase inhibitors. We hypothesize that intrinsic HIV-2 integrase variation at “secondary” HIV-1 INI-resistance sites may affect the genetic barrier to HIV-2 INI resistance. Further studies will be needed to assess INI efficacy as part of combination antiretroviral therapy in HIV-2–infected patients.     

Response of nitrifying bacterial communities to the increased thiocyanate concentration in pre-denitrification process

Changes in process performance and the nitrifying bacterial community associated with an increase of thiocyanate (SCN⁻) loading were investigated in a pre-denitrification process treating industrial wastewater. The increased SCN⁻ loading led to the concentration of total nitrogen (TN) in the final effluent, but increasing the internal recycling ratio as an operation parameter from 2 to 5 resulted in a 21% increase in TN removal efficiency. In the aerobic reactor, we found that the Nitrosomonas europaea lineage was the predominant ammonia oxidizing bacteria (AOB) and the percentages of the AOB population within the total bacteria increased from about 4.0% to 17% with increased SCN⁻ concentration. The increase of nitrite loading seemed to change the balance between Nitrospira and Nitrobacter, resulting in the high dominance of Nitrospira over Nitrobacter. Meanwhile, a Thiobacillus thioparus was suggested to be the main microorganism responsible for the SCN⁻ biodegradation observed in the system.     

A steering model of endothelial sheet migration recapitulates monolayer integrity and directed collective migration

Cells in endothelial cell monolayers maintain a tight barrier between blood and tissue, but it is not well understood how endothelial cells move within monolayers, pass each other, migrate when stimulated with growth factor, and also retain monolayer integrity. Here, we develop a quantitative steering model based on functional classes of genes identified previously in a small interfering RNA (siRNA) screen to explain how cells locally coordinate their movement to maintain monolayer integrity and collectively migrate in response to growth factor. In the model, cells autonomously migrate within the monolayer and turn in response to mechanical cues resulting from adhesive, drag, repulsive, and directed steering interactions with neighboring cells. We show that lateral-drag steering explains the local coordination of cell movement and the maintenance of monolayer integrity by allowing closure of small lesions. We further demonstrate that directional steering of cells at monolayer boundaries, combined with adhesive steering of cells behind, can explain growth factor-triggered collective migration into open space. Together, this model provides a mechanistic explanation for the observed genetic modularity and a conceptual framework for how cells can dynamically maintain sheet integrity and undergo collective directed migration.