Molecular and Functional Analysis of UDP-N-Acetylglucosamine Pyrophosphorylases from the Migratory Locust,Locusta migratoria

UDP-N-acetylglucosamine pyrophosphorylases (UAP) function in the formation of extracellular matrix by producing N-acetylglucosamine (GlcNAc) residues needed for chitin biosynthesis and protein glycosylation. Herein, we report two UAP cDNA’s derived from two different genes (LmUAP1 and LmUAP2) in the migratory locust Locusta migratoria. Both the cDNA and their deduced amino acid sequences showed about 70% identities between the two genes. Phylogenetic analysis suggests that LmUAP1 and LmUAP2 derive from a relatively recent gene duplication event. Both LmUAP1 and LmUAP2 were widely expressed in all the major tissues besides chitin-containing tissues. However, the two genes exhibited different developmental expression patterns. High expression of LmUAP1 was detected during early embryogenesis, then decreased greatly, and slowly increased before egg hatch. During nymphal development, the highest expression of LmUAP1 appeared just after molting but declined in each inter-molting period and then increased before molting to the next stage, whereas LmUAP2 was more consistently expressed throughout all these stages. When the early second- and fifth-instar nymphs (1-day-old) were injected with LmUAP1 double-stranded RNA (dsRNA), 100% mortality was observed 2 days after the injection. When the middle second- and fifth-instar nymphs (3- to 4-day-old) were injected with LmUAP1 dsRNA, 100% mortality was observed during their next molting process. In contrast, when the insects at the same stages were injected with LmUAP2 dsRNA, these insects were able to develop normally and molt to the next stage successfully. It is presumed that the lethality caused by RNAi of LmUAP1 is due to reduced chitin biosynthesis of the integument and midgut, whereas LmUAP2 is not essential for locust development at least in nymph stage. This study is expected to help better understand different functions of UAP1 and UAP2 in the locust and other insect species.     

Predicting kinetic constants of protein-protein interactions based on structural properties

Elucidating kinetic processes of protein–protein interactions (PPI) helps to understand how basic building blocks affect overall behavior of living systems. In this study, we used structure‐based properties to build predictive models for kinetic constants of PPI. A highly diverse PPI dataset, protein–protein kinetic interaction data and structures (PPKIDS), was built. PPKIDS contains 62 PPI with complex structures and kinetic constants measured experimentally. The influence of structural properties on kinetics of PPI was studied using 35 structure‐based features, describing different aspects of complex structures. Linear models for the prediction of kinetic constants were built by fitting with selected subsets of structure‐based features. The models gave correlation coefficients of 0.801, 0.732, and 0.770 for k_off, k_on, and K_d, respectively, in leave‐one‐out cross validations. The predictive models reported here use only protein complex structures as input and can be generally applied in PPI studies as well as systems biology modeling. Our study confirmed that different properties play different roles in the kinetic process of PPI. For example, k_on was affected by overall structural features of complexes, such as the composition of secondary structures, the change of translational and rotational entropy, and the electrostatic interaction; while k_off was determined by interfacial properties, such as number of contacted atom pairs per 100 Ų. This information provides useful hints for PPI design. Proteins 2010;79:720–734. © 2010 Wiley‐Liss, Inc.     

Syntheses and crystal structures of dimethyltin (IV) derivatives with 2,6-pyridinedicarboxylic acid

A novel cyclic dimethyltin complex [Me₂Sn(2,6-pdc)]₃ (1) (2,6-pdc = 2,6-pyridinedicarboxylate) was synthesized by the reaction of dimethyltin (IV) dichloride and 2,6-pyridinedicarboxylate acid in methanol under solvothermal conditions (150 °C). However, under room temperature (25 °C), we obtained a ladder complex [Me₂Sn(2,6-pdc)]₂(MeOH)₂ (2). Characterization of complexes 1 and 2 was achieved using elemental analysis, IR, ¹H, ¹³C and ¹¹⁹Sn NMR spectra and X-ray diffraction. X-ray data of 1 revealed that it was an unusual cyclic complex with a discrete cyclotrinuclear unit, in which the 12-membered cyclic cavity is almost completely planar. X-ray data of 2 showed that it was a ladder complex, in which a crystallizing methanol molecule is found in each formula unit.     

Synthesis and characterization of three group 10 complexes containing nido-carborane diphosphine: [MCl(PPh3){7,8-(PPh2)2-7,8-C2B9H10}] (M = Ni, Pd, Pt)

Three group 10 complexes containing nido-carborane diphosphine, [NiCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] (1), [PdCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 1.25CH₂Cl₂ (2) and [PtCl(PPh₃){7,8-(PPh₂)₂-7,8-C₂B₉H₁₀}] · 2.5CH₂Cl₂ (3) have been synthesized by the reactions of [M(PPh₃)₂Cl₂] (M = Ni, Pd, Pt) with closo carborane diphosphine 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ in ethanol. For complex 3, it could also be obtained under solvothermal condition. All three complexes were characterized by elemental analysis, FT-IR, ¹H and ¹³C NMR spectroscopy and X-ray structure determination. Single crystal structures show that their structures are similar to each other. In each complex, the nido [7,8-(PPh₂)₂-7,8-C₂B₉H₁₀]⁻, which resulted from the degradation of the initial closo ligand 1,2-(PPh₂)₂-1,2-C₂B₁₀H₁₀ during the reaction process, was coordinated bidentately through the P atoms to M(II) ion, and this resulted in a stable five-membered chelating ring between the bis-diphosphine ligand and the metal. The coordination mode of the metal can be described as a slightly distorted square-planar, in which the remaining two positions were occupied by one Cl⁻ and one PPh₃ group.     

A novel SNP of the <Emphasis Type="Italic">ATP1A1</Emphasis> gene is associated with heat tolerance traits in dairy cows

PCR low ionic strength single-strand conformation polymorphism (PCR-LIS-SSCP) and DNA sequencing methods were used to analyze the polymorphisms within the coding region of bovine ATP1A1 gene. A novel C/A mutation was identified at the nucleotide position 2789 of the ATP1A1 mRNA, but it was silent with respect to the amino acid sequence of the protein. The LIS-SSCP banding pattern could be divided into three kinds of genotypes, named CC, CA, and AA. Also, the association of the novel SNP of ATP1A1 gene with heat tolerance traits was studied, we found that the individuals with genotype CC showed significantly higher heat resistance than those of genotype CA (P < 0.05). Further, the mRNA levels of ATP1A1 gene in lymphocytes of peripheral blood in dairy cows among various temperature groups and genotypes were analyzed by using real-time RT-PCR. Results showed that the expression of ATP1A1 mRNA was highest in heat-stressed cows with CC genotype among the three genotypes (P < 0.01), and the ATP1A1 mRNA level at temperature 32.5°C was higher than that at optimal temperature 12.5°C in dairy cows (P < 0.01). Simultaneity, the plasma potassium (K⁺, mmol/l) and sodium (Na⁺, mmol/l) declined when the temperature dropped (P < 0.05). Our findings implied that the novel SNP here could be a potential genetic marker for anti-heat stress trait in dairy cow breeding.     

秦岭产珠子参叶的达玛烷型皂甙研究(1)

从陕西省秦岭产珠子参(Panax japonicus C.A.Meyer var.major(Burk.)Wu etFeng)的叶中分离到十个新的达玛烷型四环三萜皂甙,经光谱测定和化学降解,其中四个的化学结构分别为珠子参甙(majoroside)F_1(1)、F_2(2)、F_3(3)和F_4(4)。同时,还分离到已知的人参甙(ginsenoside)Rd(5)、Re(6)、Rg_1(7)、Rg_2(8)和F_2(9)。     

天然脂肪酸的臭氧化—氧化分解反应

本文以棉油皂脚脂肪酸为原料,用气相色谱跟踪法研究了天然脂肪酸的臭氧化一氧化分解反应,包括溶剂的选择,脂肪酸臭氧化反应温度和时间的控制以及臭氧化物氧化分解反应温度和时间的控制等。分离反应混合物,可以获得壬二酸、C_(6～9)羧酸和棕榈酸等用途广泛的化工产品。     

CIGAR‐seq,a CRISPR/Cas‐based method for unbiased screening of novel mRNA modification regulators

Cellular RNA is decorated with over 170 types of chemical modifications. Many modifications in mRNA, including m⁶A and m⁵C, have been associated with critical cellular functions under physiological and/or pathological conditions. To understand the biological functions of these modifications, it is vital to identify the regulators that modulate the modification rate. However, a high‐throughput method for unbiased screening of these regulators is so far lacking. Here, we report such a method combining pooled CRISPR screen and reporters with RNA modification readout, termed CRISPR integrated gRNA and reporter sequencing (CIGAR‐seq). Using CIGAR‐seq, we discovered NSUN6 as a novel mRNA m⁵C methyltransferase. Subsequent mRNA bisulfite sequencing in HAP1 cells without or with NSUN6 and/or NSUN2 knockout showed that NSUN6 and NSUN2 worked on non‐overlapping subsets of mRNA m⁵C sites and together contributed to almost all the m⁵C modification in mRNA. Finally, using m¹A as an example, we demonstrated that CIGAR‐seq can be easily adapted for identifying regulators of other mRNA modification.