L-histidine decarboxylase as a probe in studies on histamine

Because the Falck-Hillarp formaldehyde fluorescence method, which was superbly applied to identify catecholaminergic and serotonergic neurons, is not applicable to histamine, the first author (T.W.) developed an antibody to L-histidine decarboxylase (HDC) for identification of the histaminergic neuron system in the brain. The anti-HDC antibody was of great use for mapping the location and distribution of this histaminergic neuron system. (S)-alpha-fluoromethylhistidine, a specific and potent irreversible inhibitor of HDC, was also very useful in studies on functions of the neuron system. The activity of HDC is increased by various agents, treatments, and physiological conditions. We found new compounds that increased HDC activity (i.e., tetradecanoylphobol acetate (TPA), other tumor promoters, and staphylococcal enterotoxin A); and using mast cell-deficient mutant (W/W(v)) mice, we obtained evidence that this increase occurred in macrophages. To further characterize the mechanism of increases in HDC activity, the second author (H.O.) cloned human HDC cDNA and a human HDC gene. In studies on the regulation mechanism of the HDC gene, which is expressed only in limited types of cells such as mast cells, enterochromaffin-like cells in the stomach, cells in the tuberomammillary nucleus of the brain, and macrophages, CpG islands in the promoter region of the HDC gene were found to be demethylated in cells expressing the gene, whereas they are methylated in other cells that do not express the HDC gene. In collaboration with many other researchers, we developed HDC knockout mice. The resulting research is producing a lot of interesting findings in our laboratory as well as in others. In summary, HDC has been and will be useful in studies on functions of histamine.     

Knockout reaction mechanism studied by ~6He projectile

Knockout reaction experiment was carried out by using the 6He beams at 61.2 MeV/u impinging on a CH2 target. The α core fragments at forward angles were detected in coincidence with the recoiled protons at larger angles. From this exclusive meas urement the valence nucleon knockout mechanism and the core knockout mechanism can be distinguished by the relation be tween the polar angles of the core fragments and the recoiled protons, respectively. It is demonstrated that the core knockout mechanism may result...     

A novel molecularly imprinted method with computational simulation for the affinity isolation and knockout of baicalein from Scutellaria baicalensis

A novel molecularly imprinted polymer (MIP) was synthesized by precipitation polymerization with baicalein (BAI) as the template and used as solid‐phase extraction (SPE) adsorbent, aiming at the affinity isolation and selective knockout of BAI from Scutellaria baicalensis Georgi (SB). We used computational simulation to predict the optimal functional monomer, polymerization solvent and molar ratio of template to functional monomer. Characterization and performance tests revealed that MIP exhibited uniform spherical morphology, rapid binding kinetics, and higher adsorption capacity for BAI compared with nonimprinted polymer (NIP). The application of MIP in SPE coupled with high‐performance liquid chromatography to extract BAI from SB showed excellent recovery (94.3%) and purity (97.0%). Not only the single BAI compound, but also the BAI‐removed SB extract was obtained by one‐step process. This new method is useful for isolation and knockout of key bioactive compounds from herbal medicines. Copyright © 2015 John Wiley & Sons, Ltd.     

储存环高频剔除系统

 介绍了用于电子储存环部分填充和非均匀填充的一种实验装置,它利用储存环中电子运动所具有的横向自由振荡和束团脉冲的时间结构这一特性,采用外加激励的方法使其产生共振,从而使得储存环中部分束团中的电子丢失,形成储存环的部分填充和非均匀填充。还扼要介绍了用高频剔除系统在储存环上实现不同填充方式时的束流积累结果。     

Chemical markers’ fishing and knockout for holistic activity and interaction evaluation of the components in herbal medicines

A strategy based on chemical markers’ fishing and knockout has been proposed for holistic activity and interaction evaluation of the bioactive components in herbal medicines (HMs). It was devised to screen bioactive-compound group that represents the efficacy of HM, estimate the bioactivity contribution of each component and elucidate the interactions of multi-components. This strategy was accomplished through the following steps: (1) screen out the chemical markers (target peaks) in a HM fingerprint using online two-dimensional turbulent flow chromatography/liquid chromatography–mass spectrometry technique, (2) fish target peaks and knockout any interested peak, and (3) evaluate the bioactivities of fishing and knockout portions. After comparison of the bioactivities of samples containing different target peaks, the efficacy of target-peak group, bioactivity contribution of each compound, and the interactions of multi-components are elucidated. Using Acetylcholinesterase (AChE) and Bulbs of Lycoris radiata (L. Herit.) Herb. (BLR) as the experimental materials, four target peaks were screened out as the AChE binders. By target peaks’ fishing and knockout, combined with activity evaluation, we observed that the bioactivity of the four-peak mixture is similar with the global bioactivity of BLR extract, and there are significant suppressive actions among these four target peaks. These results indicate that this proposed strategy is a useful approach for holistic screening of bioactive-compound group and elucidation of the multi-component interactions in HM.     

Metabolism of tetrahydrobiopterin: Its relevance in monoaminergic neurons and neurological disorders

(6R)‐L‐erythro‐5,6,7,8‐tetrahydrobiopterin (BH4) is an essential cofactor for aromatic amino acid hydroxylases, such as phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), tryptophan hydroxylase, and nitric oxide synthase, which catalyze physiologically important reactions in mammals. The biosynthesis and metabolism of BH4 is usually studied mostly in the liver and only slightly in the brain, as the BH4 level in the liver is relatively high because BH4 is required for the reaction of PAH. We found that GTP (guanosine triphosphate) cyclohydrolase I, an enzyme for the biosynthesis of BH4, is a causative gene for DOPA (3,4‐dihydroxyphenylalanine)‐responsive dystonia (also called Segawa's disease), and that partial deficiency of BH4 leads to the dysfunction of the nigrostriatal dopaminergic neurons without hyperphenylalaninemia. We analyzed BH4‐deficient mice that were produced by disruption of a BH4‐synthesizing gene by a gene‐knockout technique. We found that the protein amount of TH was highly dependent on the amount of BH4, especially in nerve terminals. Our research suggests that BH4 metabolism in the brain should be different from that in the liver, and that altered metabolism of BH4 should lead to neuropsychiatric disorders including Parkinson's disease. © 2008 The Japan Chemical Journal Forum and Wiley Periodicals, Inc. Chem Rec 8: 378–385; 2008: Published online in Wiley InterScience ( www.interscience.wiley.com ) DOI 10.1002/tcr.20166     

Active Intracellular Delivery of a Cas9/sgRNA Complex Using Ultrasound‐Propelled Nanomotors

Direct and rapid intracellular delivery of a functional Cas9/sgRNA complex using ultrasound‐powered nanomotors is reported. The Cas9/sgRNA complex is loaded onto the nanomotor surface through a reversible disulfide linkage. A 5 min ultrasound treatment enables the Cas9/sgRNA‐loaded nanomotors to directly penetrate through the plasma membrane of GFP‐expressing B16F10 cells. The Cas9/sgRNA is released inside the cells to achieve highly effective GFP gene knockout. The acoustic Cas9/sgRNA‐loaded nanomotors display more than 80 % GFP knockout within 2 h of cell incubation compared to 30 % knockout using static nanowires. More impressively, the nanomotors enable highly efficient knockout with just 0.6 nm of the Cas9/sgRNA complex. This nanomotor‐based intracellular delivery method thus offers an attractive route to overcome physiological barriers for intracellular delivery of functional proteins and RNAs, thus indicating considerable promise for highly efficient therapeutic applications.     

Model-guided metabolic gene knockout of gnd for enhanced succinate production in Escherichia coli from glucose and glycerol substrates

The metabolic role of 6-phosphogluconate dehydrogenase (gnd) under anaerobic conditions with respect to succinate production in Escherichia coli remained largely unspecified. Herein we report what are to our knowledge the first metabolic gene knockout of gnd to have increased succinic acid production using both glucose and glycerol substrates in E. coli. Guided by a genome scale metabolic model, we engineered the E. coli host metabolism to enhance anaerobic production of succinic acid by deleting the gnd gene, considering its location in the boundary of oxidative and non-oxidative pentose phosphate pathway. This strategy induced either the activation of malic enzyme, causing up-regulation of phosphoenolpyruvate carboxylase (ppc) and down regulation of phosphoenolpyruvate carboxykinase (ppck) and/or prevents the decarboxylation of 6 phosphogluconate to increase the pool of glyceraldehyde-3-phosphate (GAP) that is required for the formation of phosphoenolpyruvate (PEP). This approach produced a mutant strain BMS2 with succinic acid production titers of 0.35 g l⁻¹ and 1.40 g l⁻¹ from glucose and glycerol substrates respectively. This work further clearly elucidates and informs other studies that the gnd gene, is a novel deletion target for increasing succinate production in E. coli under anaerobic condition using glucose and glycerol carbon sources. The knowledge gained in this study would help in E. coli and other microbial strains development for increasing succinate production and/or other industrial chemicals.     

GD3- and O-acetylated GD3-gangliosides in the GM2 synthase-deficient mouse brain and their immunohistochemical localization

Gangliosides in the brain of the knockout mouse deficient in the activity of β1,4 N-acetylgalactosaminyl transferase (β1,4 GalNAc-T)(GM2 synthase) consisted of nearly exclusively of GM3- and GD3-gangliosides as expected from the known substrate specificity of the enzyme and in confirmation of the initial reports from two laboratories that generated the mutant mouse experimentally. The total molar amount of gangliosides was approximately 30% higher in the mutant mouse brain than that in the wild-type brain. However, contrary to the initial reports, one-fourth of total GD3-ganglioside was O-acetylated. It reacted positively with an anti-O-acetylated GD3 monoclonal antibody and disappeared with a corresponding increase in GD3-ganglioside after mild alkaline treatment. The absence of O-acetylated GD3 in the initial reports can be explained by the saponification step included in their analytical procedures. Although quantitatively much less and identification tentative, we also detected GT3 and O-acetylated GT3. Anti-GD3 and anti-O-acetylated GD3 monoclonal antibodies gave positive reactions in the brain of mutant mouse as expected from the analytical results. Either antibody barely stained wild-type brain except for immunoreactivity of GD3 in the cerebellar Purkinje cells. The distributions of GD3 and O-acetylated GD3 in the brain of mutant mouse were similar but differential localization was noted in the cerebellar Purkinje cells and cerebral cortex.