Characterization and development of a peptide substrate-based phosphate transfer assay for the human vascular endothelial growth factor receptor-2 tyrosine kinase

Vascular endothelial growth factor (VEGF), a critical regulator in angiogenesis, exerts its angiogenic effect via binding to its receptor, VEGF receptor-2 tyrosine kinase (VEGFR2) or kinase insert domain-containing receptor (Kdr), on the surface of endothelial cells. Kdr-mediated signaling plays an important role in the proliferation, migration, differentiation, and survival of endothelial cells. Therefore, the inhibition of this signaling pathway represents a promising therapeutic approach for the discovery of novel anticancer agents by destabilizing the progression of solid tumors via abrogating tumor-induced angiogenesis. To explore Kdr as an anticancer target and further characterize the enzyme, we purified a cytoplasmic domain of human Kdr (Kdr-CD) and characterized its autophosphorylation activity. We also designed and synthesized peptides containing amino acid sequences corresponding to the autophosphorylation sites of Kdr and developed a simple, robust, high-throughput assay for measuring the phosphate transfer activity of the enzyme. This assay was validated by the experiments showing that the phosphate transfer activity of the purified Kdr-CD required Mg2+ or Mn2+ and preactivation by adenosine 5'-triphosphate (ATP) and was inhibited by known Kdr inhibitors. Using this assay, we examined effects of Mg2+ and Mn2+ on the enzyme activity; optimized the concentrations of Kdr-CD, peptide and ATP substrates, and metal ions in the assay; and determined the kinetic properties of the enzyme for the peptide and ATP as well as IC50 values of two known Kdr inhibitors. Thus, the results of these studies have validated the utilities of this assay for biochemical characterizations of the enzyme and its inhibitors. This approach of designing peptides corresponding to the autophosphorylation sites of Kdr as substrates for the enzyme has general practical implications to other kinases.     

家蝇对拟除虫菊酯抗性机理研究进展

本文概述了家蝇对拟除虫菊酯的抗性机制 ,特别是结合我国室内培育的抗拟除虫菊酯家蝇品系 (Dec R和 2Cl R) ,探讨了Na+通道、神经递质释放、ATPase、蛋白质磷酸化等与家蝇Kdr抗性的关系 ,从多方面证明了神经敏感性降低是家蝇对拟除虫菊酯产生抗性的重要机制。     

VEGFR1 (Flt1) Regulates Rab4 Recycling to Control Fibronectin Polymerization and Endothelial Vessel Branching

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear. Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports αvβ3 integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and αvβ3 integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.     

Differential expression of tumor-associated genes and altered gut microbiome with decreased Akkermansia muciniphila confer a tumor-preventive microenvironment in intestinal epithelial Pten-deficient mice

Phosphatase and tensin homolog (Pten) antagonizes PI3K-Akt signaling; therefore, Pten impairment causes tumorigenesis. However, the correlation between Pten deficiency and colon cancer has remained elusive due to numerous opposite observations. To study this correlation, we examined whether Pten deficiency in intestinal epithelial cells (IECs) induces tumorigenesis.With mucosal biopsies of human colon cancer and normal colon, Pten mRNA was evaluated by quantitative PCR. Using IEC-specific Pten knockout mice (Pten^ΔIEC/ΔIEC), we examined the mitotic activity of IECs; and Pten^ΔIEC/ΔIEC; Apc^min/+ mice were generated by combining Pten^ΔIEC/ΔIEC with Apc^min/+ mice. Tumor-associated gene was evaluated by micro-array analysis. Fecal microbiome was analyzed through 16S rRNA gene sequencing.We found that Pten mRNA level was reduced in human colon cancer relative to normal tissues. Augmented chromatids, increased Ki-67 and PCNA expression, and enhanced Akt activation were identified in IECs of Pten^ΔIEC/ΔIEC mice compared to Pten^+/+ littermate. Combining Pten^ΔIEC/ΔIEC with Apc^min/+ condition caused rapid and aggressive intestinal tumorigenesis. However, Pten^ΔIEC/ΔIEC mice did not develop any tumors. While maintaining the tumor-driving potential, these data indicated that IEC-Pten deficiency alone did not induce tumorigenesis in mice. Furthermore, the expression of tumor-promoting and tumor-suppressing genes was decreased and increased, respectively, in the intestine of Pten^ΔIEC/ΔIEC mice compared to controls. The abundance of Akkermansia muciniphila, capable of inducing chronic intestinal inflammation, was diminished in Pten^ΔIEC/ΔIEC mice compared to controls.These findings suggested that altered tumor-associated gene expression and changed gut microbiota shape a tumor-preventive microenvironment to counteract the tumor-driving potential, leading to the tumor prevention in Pten^ΔIEC/ΔIEC mice.     

Identification of mutations associated with pyrethroid resistance in the para-type sodium channel of the cat flea, Ctenocephalides felis

Knockdown resistance (kdr) to pyrethroid insecticides is caused by point mutations in the pyrethroid target site, the para-type sodium channel of nerve membranes. This most commonly involves alterations within the domain II (S4–S6) region of the channel protein where five different mutation sites have been identified across a range of insect species. To investigate the incidence of this mechanism in cat fleas, we have cloned and sequenced the IIS4–IIS6 region of the para sodium channel gene from seven laboratory flea strains. Analysis of these sequences revealed two amino acid replacements at residues previously implicated in pyrethroid resistance. One is the ‘common’ kdr mutation, a leucine to phenylalanine substitution (equivalent to L1014F of housefly) reported previously in several other insects. The other is a threonine to valine substitution (equivalent to T929V) and is a novel variant of the T929I mutation first identified in diamondback moth. The L1014F mutation was found at varying frequency in all of the laboratory flea strains, whereas the T929V mutation was found only in the highly resistant Cottontail strain. We have developed rapid PCR-based diagnostic assays for the detection of these mutations in individual cat fleas and used them to show that both L1014F and T929V are common in UK and US flea populations. This survey revealed a significant number of fleas that carry only the V929 allele indicating that co-expression with the F1014 allele is not necessary for flea viability.     

击倒抗性和钠离子通道

综述了击倒抗性与钠离子通道关系的研究进展。毒理学和电生理学的研究表明,在许多拟除虫菊酯类杀虫剂抗性昆虫中存在击倒抗性。分子遗传学研究进一步发现,击倒抗性与钠离子通道位点连锁。最近的研究表明,昆虫神经系统对拟除虫菊酯类杀虫剂敏感性下降的击倒抗性机制是钠离子通道结构基因突变。但仍有一些问题,如突变的保守性和分布,需要进一步研究、阐明。