Efficient preparation of glycoprotein hormones lacking an alpha-subunit oligosaccharide

The oligosaccharide on alpha-subunit loop 2 (alpha 2) is needed for full glycoprotein hormone efficacy. Efforts to prepare glycoprotein hormone antagonists usually involve removing the alpha 2 oligosaccharide and are hampered by its requirement for efficient heterodimer secretion from mammalian cells. Here we show that hormones lacking this oligosaccharide can be produced by treating them at low pH to dissociate the heterodimer and permitting the subunits to re-associate in the presence of peptide N-glycosidase F (PNGase F). Re-assembly of human choriogonadotropin, human follitropin, and bovine lutropin occurred rapidly and efficiently following removal of the alpha 2 oligosaccharide by PNGase F. Consequently, virtually all heterodimers formed in the presence of this enzyme lacked this oligosaccharide. These findings support the notion that heterodimer assembly in vitro occurs by a threading mechanism that is impeded by the presence of the alpha 2 oligosaccharide. This procedure should facilitate the study of glycoprotein hormone structure and function.     

The ubiquinone-binding site in NADH:ubiquinone oxidoreductase from Escherichia coli

An azido-ubiquinone derivative, 3-azido-2-methyl-5-methoxy[3H]-6-decyl-1,4-benzoquinone ([3H]azido-Q), was used to study the ubiquinone/protein interaction and to identify the ubiquinone-binding site in Escherichia coli NADH:ubiquinone oxidoreductase (complex I). The purified complex I showed no loss of activity after incubation with a 20-fold molar excess of [3H]azido-Q in the dark. Illumination of the incubated sample with long wavelength UV light for 10 min at 0 degrees C caused a 40% decrease of NADH:ubiquinone oxidoreductase activity. SDS-PAGE of the complex labeled with [3H]azido-Q followed by analysis of the radioactivity distribution among the subunits revealed that subunit NuoM was heavily labeled, suggesting that this protein houses the Q-binding site. When the [3H]azido-Q-labeled NuoM was purified from the labeled reductase by means of preparative SDS-PAGE, a 3-azido-2-methyl-5-methoxy-6-decyl-1,4-benzoquinone-linked peptide, with a retention time of 41.4 min, was obtained by high performance liquid chromatography of the protease K digest of the labeled subunit. This peptide had a partial NH2-terminal amino acid sequence of NH2-VMLIAILALV-, which corresponds to amino acid residues 184-193 of NuoM. The secondary structure prediction of NuoM using the Toppred hydropathy analysis showed that the Q-binding peptide overlaps with a proposed Q-binding motif located in the middle of the transmembrane helix 5 toward the cytoplasmic side of the membrane. Using the PHDhtm hydropathy plot, the labeled peptide is located in the transmembrane helix 4 toward the periplasmic side of the membrane.     

Improving rice yield and quality by QTL pyramiding

To facilitate marker-assisted transfer of desirable genes for improvement of yield traits, we used a set of backcross recombinant inbred lines (BRIL) derived from two elite parental lines, ‘Zhenshan97’ and ‘93-11’, to resolve a quantitative trait loci (QTL) cluster for heading date and yield-related traits in rice. Four main-effect QTL (qHD6.1, qHD6.2, qHD7, and qHD8) and four epistatic QTL affecting heading date in the BRIL were detected in two experimental trials. The major QTL (qHD8) was confirmed in three heterogeneous inbred families (HIF) that segregated for this target region, and narrowed down to a 20-kb segment in a large HIF-derived population. qHD8 was found to interact with qHD7 and had a pleiotropic effect responsible for heading date and yield components. To test usability of the identified QTL in rice improvement, we further developed near-isogenic lines (NIL) containing one or more target genes by marker-assisted transfer of ‘93-11’ alleles at qHD8, qHD7, and qHD6.1, and the GS3 gene for grain size into ‘Zhenshan97’. The pyramid line NIL(qHD8 + GS3) had higher yield potential, longer grains, and a more suitable heading date than ‘Zhenshan97’. Comparison of the NIL showed existence of epistasis between alleles at different loci and background effect on qHD8, which are very important for pyramiding of desirable alleles at the target QTL. These results will be particularly useful not only to understand the genetic basis of yield-related traits but also to improve the efficiency of marker-assisted selection for favorable loci in rice breeding programs.     

松油烯-４-醇对粘虫幼虫的生物活性

测定了杀虫植物砂地柏Sabina vulgaris Ant.的精油中主杀虫成分-松油烯-4-醇（terpinen 4.01）对粘虫Mythimna separata Walker幼虫的生物活性。结果表明,松油烯- 4-醇对粘虫主要表现为熏蒸作用,对粘虫3龄幼虫24 h的熏蒸LC₅₀为5.3473 μL/L ;还具一定触杀作用,对粘虫4龄幼虫24 h的LD₅₀为147.8 μg/虫。试虫的中毒症状可明显地分为兴奋、痉挛、麻痹和死亡4个阶段,而麻痹的部分试虫有复苏现象。可明显抑制Na⁺ ,K⁺ATP酶的活性,在兴奋期、痉挛期、麻痹期和复苏期,抑制率介于21.28%～34.92% 之间。离体条件下对Na⁺,K⁺ATP酶的I₅₀为133.75 μg·mL^-1;对AChE活性有一定的影响;对酯酶,在兴奋期,酶活力为对照的7.0%,在麻痹期则为对照的1.33倍,而复苏期试虫的酯酶活力与对照相当。