Selection of trkB-binding peptides from a phage-displayed random peptide library

Brain-derived neurotrophic factor (BDNF) shows potential in the treatment of neurodegenerative diseases, but the therapeutic application of BDNF has been greatly limited because it is too large in molecular size to permeate blood-brain barrier. To develop low-molecular-weight BDNF-like peptides, we selected a phage-displayed random peptide library using trkB expressed on NIH 3T3 cells as target in the study. With the strategy of peptide library incubation with NIH 3T3 cells and competitive elution with 1 μg/mL of BDNF in the last round of selection, the specific phages able to bind to the natural conformation of trkB and antagonize BDNF binding to trkB were enriched effectively. Five trkB-binding peptides were obtained, in which a core sequence of CRA/TXΦXXΦXXC (X represents the random amino acids, Φ represents T, L or I) was identified. The BDNF-like activity of these five peptides displayed on phages was not observed, though all of them antagonized the activity of BDNF in a dose-dependent manner. Similar results were obtained with the synthetic peptide of C1 clone, indicating that the 5 phage-derived peptides were trkB antagonists. These low-molecular-weight antagonists of trkB may be of potential application in the treatment of neuroblastoma and chronic pain. Meanwhile, the obtained core sequence also could be used as the base to construct the secondary phage-displayed peptide library for further development of small peptides mimicking BDNF activity.     

琥珀酸脱氢酶或琥珀酰辅酶A合成酶缺失促进大肠杆菌积累5-氨基乙酰丙酸

5-氨基乙酰丙酸 (ALA) 是生物体内四吡咯类化合物的合成前体,在农业及医药领域应用广泛,是极具开发价值的高附加值生物基化学品。目前利用外源C4途径的重组大肠杆菌发酵生产ALA的研究主要利用LB培养基并添加葡萄糖和琥珀酸、甘氨酸等合成前体,成本较高。琥珀酸在C4途径中以琥珀酰辅酶A的形式直接参与ALA的合成。文中在以葡萄糖为主要碳源的无机盐培养基中研究了琥珀酰辅酶A下游代谢途径琥珀酸脱氢酶编码基因sdhAB和琥珀酰辅酶A合成酶编码基因sucCD缺失对ALA积累的影响。与仅表达异源ALA合成酶的对照菌株相比,sdhAB和sucCD缺失菌株ALA的产量分别提高了25.59%和12.40%,且ALA的积累不依赖于琥珀酸的添加和LB培养基的使用,从而大幅降低了生产成本,显示出良好的工业应用前景。     

Comparison and Effects of Acute Lamotrigine Treatment on Extracellular Excitatory Amino Acids in the Hippocampus of PTZ-Kindled Epileptic and PTZ-Induced Status Epilepticus Rats

In this communication, the effect of acute treatment with lamotrigine (LTG) was investigated on release of main excitatory amino acids (EAA) such as glutamate (Glu) and aspartate (Asp) in the hippocampus of pentylenetetrazol (PTZ)-induced and PTZ-kindled freely moving rats using micro dialysis. The results show that, levels of Glu and Asp significantly increased in the rat hippocampus during the seizure/interical periods for PTZ-status epilepticus (SE) and PTZ-kindled epileptic (EP) rats. The levels of Glu and Asp increased more in EP rat hippocampus than in SE rat hippocampus. After administration of 20 mg/kg LTG, the levels of Glu and Asp significantly decreased in the SE and EP rat hippocampus. The results indicate that: (a) excitability of the PTZ-kindled epileptogenic model is higher than that of the status epilepticus model; (b) the modulation of LTG on the EAA neurotransmitters certainly plays an important role in antiepileptic efficacy, especially in PTZ-kindled epileptic model where the release of EAA was influenced more markedly by acute application of 20 mg/kg LTG.     

Developmental Lead Exposure and Two-Way Active Avoidance Training Alter the Distribution of Protein Kinase C Activity in the Rat Hippocampus

Long-term exposure to a low level of lead is associated with learning deficits. Several types of learning have been correlated to hippocampal protein kinase C (PKC) activation. This study was designed to determine if there is a correlation between the effects of lead on hippocampal PKC activation and those on learning performance. Rats were exposed to 0.2% (w/v) lead acetate at different developmental stages including a maternally exposed group, a postweaning exposed group, and a continuously exposed group. The continuously lead exposed rats tended to avoid less frequently and not respond more frequently in two-way active avoidance training than did controls. This training process was associated with translocation of hippocampal PKC activity from cytosol to membrane. Two-way analysis of variance of data indicates that there is a significant training and lead treatment interaction in the ratio of membrane to cytosolic PKC activity (F_3,32 = 3.013; p = 0.044). The interaction is attributable to the absence of the training-induced PKC translocation in the continuously lead exposed rats. In addition, no significant changes were observed in learning performance and training-induced hippocampal PKC activation after maternal and postweaning lead exposure. Continuous and longer duration of lead exposure appears to affect the learning performance and hippocampal PKC activation. These data suggest that a change in the activation of hippocampal PKC may be involved in the lead-induced deficit in learning.     

Whole‐brain microcirculation detection after ischemic stroke based on swept‐source optical coherence tomography

The occurrence and development of ischemic stroke are closely related to cerebral blood flow. Real‐time monitoring of cerebral perfusion level is very useful for understanding the mechanisms of the disease. A wide field of view (FOV) is conducive to capturing lesions and observing the progression of the disease. In this paper, we attempt to monitor the whole‐brain microcirculation in middle cerebral artery occlusion (MCAO) rats over time using a wide FOV swept‐source OCT (SS‐OCT) system. A constrained image registration algorithm is used to remove motion artifacts that are prone to occur in a wide FOV angiography. During ischemia, cerebral perfusion levels in the left and right hemispheres, as well as in the whole brain were quantified and compared. Changes in the shape and location of blood vessels were also recorded. The results showed that the trend in cerebral perfusion levels of both hemispheres was highly consistent during MCAO, and the position of the blood vessels varied over time. This work will provide new insights of ischemic stroke and is helpful to assess the effectiveness of potential treatment strategies.      

一株假单胞菌(Pseudomonas sp.)L-1菌株γ-丁基甜菜碱羟化酶基因bbh 的克隆、表达及酶学性质

【目的】γ-丁基甜菜碱羟化酶是生物体内合成L-肉碱的关键酶。从假单胞菌(Pseudomonas sp.)L-1中克隆γ-丁基甜菜碱羟化酶基因,实现其在大肠杆菌(Escherichia coli)中的高效表达,并对表达产物进行酶学性质分析,为生物转化生产L-肉碱奠定基础。【方法】通过PCR克隆γ-丁基甜菜碱羟化酶基因,并将其开放阅读框(ORF)克隆至融合表达载体pET-15b;表达产物经His.Bind Resin纯化后对BBH进行酶学性质及三维空间结构分析;并以静止细胞进行L-肉碱的转化。【结果】成功地克隆了一个γ-丁基甜菜碱羟化酶基因bbh(GenBank:JQ250036),并实现了其在E.coli中的高效表达。融合蛋白以同源二聚体的形式存在,单个亚基的分子量约46.5 kDa,最适反应温度为30℃,最适反应pH为7.5。该酶在45℃以下稳定。在pH6.0时该酶有最高的pH稳定性。以表达bbh基因的重组大肠杆菌静止细胞转化L-肉碱,L-肉碱产量可达12.7mmol/L。【结论】Pseudomonas sp.L-1γ-丁基甜菜碱羟化酶与现有报道的bbh基因有较大的差异。由该基因表达的γ-丁基甜菜碱羟化酶能有效地转化γ-丁基甜菜碱生成L-肉碱。本研究不仅丰富了γ-丁基甜菜碱羟化酶基因资源,而且为L-肉碱的生物转化提供了一种新的转化方案。     

Activation of NF-κB in CD8+ dendritic cells Ex Vivo by the γ134.5 null mutant correlates with immunity against herpes simplex virus 1

The γ₁34.5 protein of herpes simplex viruses (HSV) is essential for virulence. Accordingly, an HSV mutant lacking γ₁34.5 is attenuated in vivo. Despite its vaccine potential, the mechanism by which the γ₁34.5 null mutant triggers protective immunity is unknown. In this report we show that vaccination with the γ₁34.5 null mutant protects against lethal challenge from wild-type virus via IκB kinase in dendritic cells (DCs), which sense virus-associated molecular patterns. Unlike mock-treated DCs, DCs primed with the γ₁34.5 null mutant ex vivo mediate resistance to wild-type HSV after adoptive transfer into naïve mice. Furthermore, the γ₁34.5 null mutant activates IκB kinase, which facilitates p65/RelA phosphorylation and nuclear translocation, resulting in DC maturation. While unable to produce infectious virus in DCs, this mutant virus expresses early and late genes. In its abortive infection, the γ₁34.5 null mutant induces protective immunity more effectively in CD8⁺ DCs than in CD8⁻ DCs. This is mirrored by a higher level of interleukin-6 (IL-6) and IL-12 secretion by CD8⁺ DCs than CD8⁻ DCs. Remarkably, inhibition of p65/RelA phosphorylation or nuclear translocation in CD8⁺ DCs disrupts protective immunity. These results suggest that engagement of the γ₁34.5 null mutant with CD8⁺ DCs elicits innate immunity to activate NF-κB, which translates into protective immunity.     

The effects of garlic-derived sulfur compounds on cell proliferation, caspase 3 activity, thiol levels and anaerobic sulfur metabolism in human hepatoblastoma HepG2 cells

The aim of the present studies was to determine whether the mechanism of biological action of garlic-derived sulfur compounds in human hepatoma (HepG2) cells can be dependent on the presence of labile sulfane sulfur in their molecules. We investigated the effect of allyl sulfides from garlic: monosulfide, disulfide and trisulfide on cell proliferation and viability, caspase 3 activity and hydrogen peroxide (H(2)O(2)) production in HepG2 cells. In parallel, we also examined the influence of the previously mentioned compounds on the levels of thiols, glutathione, cysteine and cysteinyl-glycine, and on the level of sulfane sulfur and the activity of its metabolic enzymes: rhodanese, 3-mercaptopyruvate sulfurtransferase and cystathionase. Among the compounds under study, diallyl trisulfide (DATS), a sulfane sulfur-containing compound, showed the highest biological activity in HepG2 cells. This compound increased the H(2)O(2) formation, lowered the thiol level and produced the strongest inhibition of cell proliferation and the greatest induction of caspase 3 activity in HepG2 cells. DATS did not affect the activity of sulfurtransferases and lowered sulfane sulfur level in HepG2 cells. It appears that sulfane sulfur containing DATS can be bioreduced in cancer cells to hydroperthiol that leads to H(2)O(2) generation, thereby influencing transmission of signals regulating cell proliferation and apoptosis.     

Genetic diversity and genetic structure of different populations of the endangered species Davidia involucrata in China detected by inter-simple sequence repeat analysis

Dove tree (Davidia involucrate) is a Tertiary relic species endemic to China and is reputed to be a ‘living fossil’ in the plant kingdom. Genetic diversity and genetic structure of this species were analyzed for its conservation and management, using inter-simple sequence repeat (ISSR) data obtained from eight populations distributed throughout seven provinces of China. A relatively high level of genetic diversity, at both population and species levels, was detected using the POPGENE software. Analysis of molecular variance (AMOVA) revealed a moderate level of among-population variation (i.e., 33.21%). The genetic structure of dove tree was closely consistent with their isolated topographical distribution region based on the results of the STRUCTURE, POPGENE-UPGMA and PCA analysis. It is postulated that the relatively high level of genetic diversity has been maintained because of: (a) the original wild geographical distribution, (b) propagation through outcrossing seeds or root suckers, (c) the longevity of individuals and (d) the relatively little human disturbance. Genetic drift and restricted gene are important factors affecting genetic differentiation. There was no significant correlation between geographical distances and a pairwise comparison with genetic distances, as analyzed by the Mantel test, but the clustering result of genetic diversity was consistent with their isolated topographical distribution regions. Thus, maintaining the stable special habitats associated with this species is recommended for the in situ conservation. Furthermore, it is important to develop an effective seed germination system for the maintenance of an ex situ conservation pool of the germplasm resources.