Design, synthesis, insecticidal activity and 3D-QSR study for novel trifluoromethyl pyridine derivatives containing an 1,3,4-oxadiazole moiety.

A series of trifluoromethyl pyridine derivatives containing 1,3,4-oxadiazole moiety was designed, synthesized and bio-assayed for their insecticidal activity. The result of bio-assays indicated the synthesized compounds exhibited good insecticidal activity against Mythimna separata and Plutella xylostella, most of the title compounds show 100% insecticidal activity at 500 mg L-1 and >80% activity at 250 mg L-1 against the two pests. Compounds E18 and E27 showed LC50 values of 38.5 and 30.8 mg L-1 against Mythimna separata, respectively, which were close to that of avermectin (29.6 mg L-1); compounds E5, E6, E9, E10, E15, E25, E26, and E27 showed 100% activity at 250 mg L-1, which were better than chlorpyrifos (87%). CoMFA and CoMSIA models with good predictability were proposed, which revealed the electron-withdrawing groups with an appropriate bulk at 2- and 4-positions of benzene ring could enhance insecticidal activity.

Intracellular localization and association of MHC class I with porcine invariant chain.

The objective was to investigate the intracellular localization and association of pig major histocompatibility complex (MHC) class I subunits with invariant chain (Ii). Pig MHC class I subunit cDNAs were cloned by RT-PCR and eukaryotic expression plasmids of α and ß2m were constructed with fusions to red or enhanced green fluorescent protein (pDsRed2-N1-α, pEGFP-N1-α, pDsRed2-N1-ß2m, and pEGFP-N1-ß2m). A pig Ii mutant with a deleted CLIP region (DCLIP-Ii) was constructed by overlap extension PCR. Wild-type Ii and mutant Ii were cloned into pEGFP-C1 (pEGFP-C1-Ii, pEGFP-C1-DCLIP-Ii). The recombinant plasmids of MHC I subunits and pEGFP-C1-Ii (pEGFP-C1-ΔCLIP-Ii) were transiently cotransfected into COS-7 cells with Lipofectamine 2000. Immunofluorescence microscopy was performed to detect expression and intracellular localization of Ii and MHC I subunits, and immunoprecipitation was used to analyze their association. Our results indicated that pig Ii associates with integrated MHC I subunits to form oligomers, but cannot associate with single MHC I subunits. Furthermore, deletion of the Ii CLIP sequence blocks association with integrated MHC I subunits. Thus, pig Ii cannot associate with a single MHC I molecule, the α or ß2m chain, but Ii and the integrated MHC I molecule can form complexes that colocalize in the endomembranes of COS-7 cells. The Ii of CLIP plays a key role in assembly of Ii and MHC I.

Molecular cloning and site-directed mutagenesis of leucine-based sorting motifs of the porcine invariant chain.

Invariant chain (Ii) is a transmembrane protein that associates with MHC class II molecules in the endoplasmic reticulum. The cytoplasmic tail of Ii contains two leucine residues able to direct Ii to the endocytic pathway. We obtained the pig Ii gene by RT-PCR. Mutated Ii was prepared via site directed mutagenesis by the PCR Megaprimer method to study the effect of the two leucines on the localization of pig Ii. These mutated fragments were ligated to the vector pmCherry-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine(TM) 2000. Fluorescence of fusion proteins (mCherry-Ii) was observed with a fluorescent microscope. Amino acid sequence alignment showed that pig Ii has domains similar to those seen in other mammalian Ii, including the cytoplasmic, transmembrane, class II-associated Ii-derived peptide, and trimerization domains. Based on observations with the fluorescent microscope, we found that two leucine-based motifs are required for pig Ii intracellular localization, and that both motifs independently mediate this function in Ii.

Effects of replacing soybean meal with fermented rapeseed meal on performance, serum biochemical variables and intestinal morphology of broilers.

This trial was performed to study the effects of replacing soybean meal (SBM) with fermented rapeseed meal (RSM) on growth performance, serum biochemistry variable and intestinal morphology of broilers. A total of 640 d-old Arbor Acres broiler chicks were randomly allocated to 4 dietary treatments, 4 pens per treatment and 40 birds per pen for a 6-wk feeding trial. In the four treatment groups, fermented RSM replaced soybean meal at 0, 5, 10, and 15%, respectively. On 21 d and 42 d, two birds from each pen were randomly selected and slaughtered. Blood samples and sections of duodenum, jejunum, and ileum were collected for measurement of serum biochemical variables and intestinal morphology, respectively. Results showed that body weight gain (BWG) and feed conversion (FC) were significantly (p<0.01) poorer for birds fed the 15% fermented RSM diet than those fed with 0, 5 and 10% fermented RSM diets during all periods. Compared with 0 and 5% fermented RSM groups, IgG content in the serum of birds in 10 and 15% fermented RSM groups was improved (p<0.01) urea nitrogen content of serum was reduced (p<0.01) during both growing and finishing periods. However, IgM, phosphorus and calcium levels increased (p<0.05) only during the growing period. Increased (p<0.05) villus height was observed in the duodenum and jejunum of broilers fed the diet with 10% fermented RSM. In addition, villus height to crypt depth ratio in the jejunum was significantly higher (p<0.01) for birds fed the diet with 10% fermented RSM than for those fed diets with 0, 5 and 15% fermented RSM. The present results suggest that RSM fermented with Lactobacillus fermentum and Bacillus subtilis is a promising alternative protein source and that it could be safely used replace up to 10% SBM in broiler diets.

The intracellular localization and oligomerization of chicken invariant chain with major histocompatibility complex class II subunits.

Invariant chain (Ii) binds to MHC class II (MHCII) to assemble a nonamer in the endoplasmic reticulum. Major histocompatibility complex class II-associated Ii peptide (CLIP) that occupies the peptide binding groove of MHCII prevents MHCII molecules from loading with endogenous antigens. We used the green or red fluorescent protein-fused Ii or MHCII subunits to detect the intracellular localization and oligomerization of chicken Ii with single chicken MHCII subunits. Our results indicated that chicken Ii associates with single MHCII subunits and formed oligomers with MHCII subunits. The Ii mutant with a deleted CLIP sequence blocks the association with single MHCII subunits, but exchanging CLIP with the Newcastle disease virus F(343) epitope restores this association. Thus, MHCII polymer assembly is not blocked as long as the basic steric molecular structure of Ii is maintained, and different binding models exist in different species or MHCII isotypes.

The effect of site-directed mutagenesis of the ambient amino acids of leucine-based sorting motifs on the localization of chicken invariant chain.

Two Leu residues and their ambient amino acid residues are known to exist in the cytosolic tail of chicken invariant chain (Ii), and these play an important role as motifs in mediating the sorting endocytic pathway. We performed 20 mutations via site-directed mutagenesis by the PCR megaprimer method to study the effect of some ambient amino residues of both Leu on the localization of chicken Ii. These mutated fragments were ligated to the vector pEGFP-C1. The recombinant plasmids were transiently transfected into COS-7 cells with Lipofectamine 2000. Furthermore, the fluorescence of located fusion proteins (green fluorescent protein-Ii) was observed with a fluorescence microscope. Our results indicated that 2 Leu-based motifs are required for chicken Ii intracellular localization, and both motifs independently mediate this function of the Ii. The other amino acid residues surrounding both Leu also influence Ii-induced endosomal vacuolation. In addition, we found that Pro19, which is near the Val17-Leu18 motif, was a key residue for chicken Ii intracellular localization. Not only is it critical for endocytic targeting to each Leu, but its unique mutation can also result in altering the function of chicken Ii.

Anatomic microstructure of the upper eyelid in the Oriental double eyelid.

The anatomic differences in the microstructure of the upper eyelid between the double eyelid and the nondouble eyelid are compared to determine the mechanism of double eyelid formation. Tissue from the upper eyelids of normal adult women was categorized into three groups: in one group, the double eyelid was formed primarily (at birth); in a second group, the double eyelid was formed gradually; and those in a third group had nondouble eyelids. A total of 56 eyelids were studied using electron microscopy and light microscopy. The results indicated that there is a significant difference between the three groups using scanning electron microscopy. In the upper eyelid of the double eyelid, bunched fibers of levator aponeurosis penetrate through orbicularis muscle to fuse with the skin in palpebral sulcus. This structure was not observed in the group with nondouble eyelids. However, when using light microscopy, this disparity was not observed. It was concluded that a fiber-linked structure between eyelid skin and levator aponeurosis is essential for the formation of the double eyelid.