High-performance liquid chromatographic method for the determination of a novel thymidylate synthase inhibitor, AG 331, in human serum

AG 331 is a novel thymidylate synthase inhibitor currently in Phase I clinical trial. To determine the pharmacokinetic parameters of AG 331 in human subjects, a suitable analytical method was developed using high-performance liquid chromatography. Serum and urine samples were prepared using both solid-phase extraction and solvent extraction. Either 4,4′-diaminodiphenyl sulfone or benz[cd]indole-2(1H)-one were used as internal standards for the method. A reversed-phase C₁₈ analytical column completely resolved the drug and internal standard peaks from non-specific substances present in biological matrix. The method was validated for precision, accuracy, and reproducibility in serum and was linear over a concentration range of 50–2000 ng/ml, with a limit of detection of 20.0 ng/ml and a quantifiable limit of 50 ng/ml.     

Midkine noncanonically suppresses AMPK activation through disrupting the LKB1-STRAD-Mo25 complex

Midkine (MDK), a secreted growth factor, regulates signal transduction and cancer progression by interacting with receptors, and it can be internalized into the cytoplasm by endocytosis. However, its intracellular function and signaling regulation remain unclear. Here, we show that intracellular MDK interacts with LKB1 and STRAD to disrupt the LKB1-STRAD-Mo25 complex. Consequently, MDK decreases the activity of LKB1 to dampen both the basal and stress-induced activation of AMPK by glucose starvation or treatment of 2-DG. We also found that MDK accelerates cancer cell proliferation by inhibiting the activation of the LKB1-AMPK axis. In human cancers, compared to other well-known growth factors, MDK expression is most significantly upregulated in cancers, especially in liver, kidney and breast cancers, correlating with clinical outcomes and inversely correlating with phosphorylated AMPK levels. Our study elucidates an inhibitory mechanism for AMPK activation, which is mediated by the intracellular MDK through disrupting the LKB1-STRAD-Mo25 complex.Subject terms: Cancer, Oncogenes     

Dopamine D3 receptor signaling alleviates mouse rheumatoid arthritis by promoting Toll-like receptor 4 degradation in mast cells

Dopamine receptors are involved in several immunological diseases. We previously found that dopamine D3 receptor (D3R) on mast cells showed a high correlation with disease activity in patients with rheumatoid arthritis, but the mechanism remains largely elusive. In this study, a murine collagen-induced arthritis (CIA) model was employed in both DBA/1 mice and D3R knockout mice. Here, we revealed that D3R-deficient mice developed more severe arthritis than wild-type mice. D3R suppressed mast cell activation in vivo and in vitro via a Toll-like receptor 4 (TLR4)-dependent pathway. Importantly, D3R promoted LC3 conversion to accelerate ubiquitin-labeled TLR4 degradation. Mechanistically, D3R inhibited mTOR and AKT phosphorylation while enhancing AMPK phosphorylation in activated mast cells, which was followed by autophagy-dependent protein degradation of TLR4. In total, we found that D3R on mast cells alleviated inflammation in mouse rheumatoid arthritis through the mTOR/AKT/AMPK-LC3-ubiquitin-TLR4 signaling axis. These findings identify a protective function of D3R against excessive inflammation in mast cells, expanding significant insight into the pathogenesis of rheumatoid arthritis and providing a possible target for future treatment.Subject terms: Immunological disorders, Rheumatic diseases     

Interaction between calcium-free calmodulin and IQ motif of neurogranin studied by nuclear magnetic resonance spectroscopy

The interaction of Ca(2+)-free calmodulin (apoCaM) with the IQ motif corresponding to the calmodulin-binding domain of neurogranin has been studied by nuclear magnetic resonance (NMR) methods. The NMR spectra of uncomplexed apoCaM and apoCaM in complex with the IQ motif recorded at 750 MHz were studied and the backbone assignments of the protein in both forms were obtained by triple-resonance multidimensional NMR experiments. Chemical shift perturbations were used to map the binding surfaces. Only a single set of resonances was observed throughout the titration, indicating that the binding interaction is under fast exchange. Analysis of chemical shift changes indicates that (a) the main interaction and conformational changes occur in the C-terminal domain of calmodulin and (b) linker-1 (residues 40-44) between EF-1 and EF-2, linker-3 (residues 112-117) between EF-3 and EF-4, and the end of the alpha-helix H (residues 145-148) may be involved in the binding process. The dissociation constant (K(d)), estimated by fitting the chemical shift changes against the IQ peptide concentration, ranged from about 1.2 x 10(-5) to 8.8 x 10(-5) M. This result demonstrates that the interaction falls into the weak binding regime.     

Heterologous expression of a mammalian protein tyrosine phosphatase gene in Leishmania: effect on differentiation

Leishmania is a protozoan pathogen which is transmitted to humans through the bite of an infected sandfly. This infection results in a spectrum of diseases throughout the developing world, collectively known as leishmaniasis. During its life cycle, Leishmania differentiates from the promastigote stage in the sandfly vector into the amastigote stage in the mammalian host where it multiplies exclusively in macrophage phagolysosomes. Although differentiation of Leishmania is essential for its survival and pathogenesis in the mammalian host, this process is poorly understood. In higher eukaryotic cells, protein tyrosine phosphorylation plays a central role in cell proliferation, differentiation and overall function. We have therefore investigated the role of protein tyrosine phosphorylation in Leishmania differentiation by undertaking complementary approaches to mediate protein tyrosine dephosphorylation in vivo. In the present study, L. donovani were engineered to express a mammalian protein tyrosine phosphatase, or were treated with inhibitors of protein tyrosine kinases, and the resulting phenotype was examined. Both approaches resulted in a partial differentiation from promastigotes to amastigotes including the expression of the amastigote specific A2 protein, morphological change and increased virulence. These data provide support for the involvement of tyrosine phosphorylation in the differentiation of Leishmania.     

氯氟氰菊酯与阿维菌素增效混配制剂对美洲斑潜蝇的防治效果

低毒化学杀虫剂氯氟氰菊酯与生物源农药阿维菌素混配,通过其对美洲斑潜蝇室内毒力实验,测定共毒系数CTC为161～232,处于明显增效范围内。据此确定最佳配比,配制此增效混剂2%渗透型可湿性粉剂。在北京、山东两地防治美洲斑潜蝇幼虫的田间试验表明药效优良,制剂用量50 g/667m²药后3、7、11天两地区校正防效分别为85.26%～90.76%和86.74%～94.02%,制剂用量25g/667m2两地区相应防效分别为75.28%～85.17%、79.96%～88.68%。该增效混剂防治斑潜蝇速效性和持效性皆佳,成本有所下降,使用可湿粉与乳油相比较,可减少投放入环境的化学品数量,以减少环境污染。     

Methylene Blue as a Cerebral Metabolic and Hemodynamic Enhancer

By restoring mitochondrial function, methylene blue (MB) is an effective neuroprotectant in many neurological disorders (e.g., Parkinson’s and Alzheimer’s diseases). MB has also been proposed as a brain metabolic enhancer because of its action on mitochondrial cytochrome c oxidase. We used in vitro and in vivo approaches to determine how MB affects brain metabolism and hemodynamics. For in vitro, we evaluated the effect of MB on brain mitochondrial function, oxygen consumption, and glucose uptake. For in vivo, we applied neuroimaging and intravenous measurements to determine MB’s effect on glucose uptake, cerebral blood flow (CBF), and cerebral metabolic rate of oxygen (CMRO₂) under normoxic and hypoxic conditions in rats. MB significantly increases mitochondrial complex I–III activity in isolated mitochondria and enhances oxygen consumption and glucose uptake in HT-22 cells. Using positron emission tomography and magnetic resonance imaging (MRI), we observed significant increases in brain glucose uptake, CBF, and CMRO₂ under both normoxic and hypoxic conditions. Further, MRI revealed that MB dramatically increased CBF in the hippocampus and in the cingulate, motor, and frontoparietal cortices, areas of the brain affected by Alzheimer’s and Parkinson’s diseases. Our results suggest that MB can enhance brain metabolism and hemodynamics, and multimetric neuroimaging systems offer a noninvasive, nondestructive way to evaluate treatment efficacy.     

BAK1, an Arabidopsis LRR receptor-like protein kinase,interacts with BRI1 and modulates brassinosteroid signaling

Brassinosteroids regulate plant growth and development through a protein complex that includes the leucine-rich repeat receptor-like protein kinase (LRR-RLK) brassinosteroid-insensitive 1 (BRI1). Activation tagging was used to identify a dominant genetic suppressor of bri1, bak1-1D (bri1-associated receptor kinase 1-1Dominant), which encodes an LRR-RLK, distinct from BRI1. Overexpression of BAK1 results in elongated organ phenotypes, while a null allele of BAK1 displays a semidwarfed phenotype and has reduced sensitivity to brassinosteroids (BRs). BAK1 is a serine/threonine protein kinase, and BRI1 and BAK1 interact in vitro and in vivo. Expression of a dominant-negative mutant allele of BAK1 causes a severe dwarf phenotype, resembling the phenotype of null bri1 alleles. These results indicate BAK1 is a component of BR signaling.     

小麦高分子量麦谷蛋白亚基分离方法的研究

小麦高分子量麦谷蛋白亚基（ＨＭＷ－ＧＳ）与小麦面包烘烤质量和面粉的加工特性密切相关,ＳＤＳ－ＰＡＧＥ是其常用的分离方法之一。ＳＤＳ－ＰＡＧＥ方法一般分为２类：第一类采用１１％和５％浓度的胶,后者用于分离２亚基和２＾＊亚基,该种方法常使用碱性提取液,需要２次电泳过程,且在５％浓度的胶中ＨＭＷ－ＧＳ易于和麦醇蛋白混淆;另外一类ＳＤＳ－ＰＡＧＥ采用梯度胶,配合使用银染方法,制梯度胶则使用梯度仪及磁力搅拌