Human brain aminopeptidase A: biochemical properties and distribution in brain nuclei

Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.     

Changes in social environment induce higher emotional disturbances than changes in physical environment in quail

In intensive rearing, birds are exposed to frequent changes in both their social and physical environments, and this can have an impact on animal welfare under commercial conditions. The aim of the present study was to compare the emotional responses induced by social and non-social changes and to study the influence of the familiarity on these responses.Twenty-two female quail were first reared with a ball in 15–20-individual groups for 3 weeks from hatching. Then, each experimental subject was allocated either a familiar congener (CONG) or a familiar object (BALL). At 6 weeks of age the birds were fitted with a telemetric device to collect motor and cardiac activities. After that, each subject was exposed first to a separation from its cage-mate/familiar object, and secondly to the (re-)introduction of either the cage-mate/familiar object or of an unknown conspecific/another ball. Emotional responses were assessed through behavioural reactions and heart rate variability.Before being separated, both BALL and CONG quail showed stereotyped pacing that was more pronounced in BALL quail. BALL quail were not affected by the ball withdrawal, unlike CONG quail which reacted to the separation from their cage-mate by reducing activity and exploratory behaviour (P < 0.05). After the re-introduction, BALL quail remained closer to their ball and CONG quail spent more time with “stretched necks” than before the separation (P < 0.05). Surprisingly, CONG quail showed less stereotyped pacing when their partner was removed and recovered the initial level of stereotyped behaviour when their cage-mate was re-introduced (P < 0.01). Likewise, CONG quail also recovered the initial values of heart rate after the re-introduction of their cage-mate, indicating an increase in sympathetic activity compared to the isolation period. When the unfamiliar congener or ball was introduced in their cage, BALL quail pecked the unknown ball more than the familiar ball and had more contact with it (P < 0.05) and CONG quail had more contact with the unknown congener than with the cage-mate (P < 0.05).In conclusion, the quail appeared to experience a negative affective state before being separated, and there was no clear evidence of negative emotion in quail in response to the separation from either a social partner or an inanimate object. Nevertheless, the emotional responses of the quail in reaction to the (re-)introduction was influenced by the familiarity of the congener or the ball. Finally, changes in the quail's social environment induced more behavioural and cardiac modifications than changes in its non-social environment.     

A Model System for the Study of the Release of Luteinizing Hormone-Releasing Hormone from Isolated Storage Granules

Abstract: We have developed an in vitro system for the study of the release of luteinizing hormone-releasing hormone (LH-RH) from its storage granules. In this system, homogenates of hypothalamic tissue are subjected to hypoosmotic shock, and the LH-RH-containing granules are isolated by means of differential centrifugation. The isolated granules are then incubated in a buffered medium, and the incubation is terminated by passing the incubation mixture through LH-RH affinity columns. The LH-RH associated with the granules passes freely through the columns, whereas the LH-RH released into the medium binds to the columns and is subsequently eluted with an acid solution. LH-RH is quantified by radioimmunoassay (RIA). We tested the effects of various concentrations of KCl on LH-RH release, which was found to be dependent on the concentration of KCl in the medium over the range 40–160 mM. We then studied the effects of pH on the release of LH-RH. Incubation of granules at pH 7.8 in the presence of 160 mM-KC1 resulted in the release from the granules of 14% of the stored LH-RH, whereas incubation at pH 6.2 resulted in the release of approximately 30% of the LH-RH. In addition, granules were incubated at pH 7.8 with MgATP and KCl. MgATP elicited a marked release of LH-RH that was approximately twice that seen in the absence of MgATP. In summary, in this in vitro system, granules containing LH-RH are stable under defined biochemical conditions, and LH-RH release from these granules is stimulated by ions and MgATP.     

Magnetic nanoparticles with surface modification enhanced gene delivery of HVJ-E vector

To enter the realm of human gene therapy, a novel drug delivery system is required for efficient delivery of small molecules with high safety for clinical usage. We have developed a unique vector HVJ-E (hemagglutinating virus of Japan-envelope) that can rapidly transfer plasmid DNA, oligonucleotide, and protein into cells by cell-fusion. In this study, we associated HVJ-E with magnetic nanoparticles, which can potentially enhance its transfection efficiency in the presence of a magnetic force. Magnetic nanoparticles, such as maghemite, with an average size of 29 nm, can be regulated by a magnetic force and basically consist of oxidized Fe which is commonly used as a supplement for the treatment of anemia. A mixture of magnetite particles with protamine sulfate, which gives a cationic surface charge on the maghemite particles, significantly enhanced the transfection efficiency in an in vitro cell culture system based on HVJ-E technology, resulting in a reduction in the required titer of HVJ. Addition of magnetic nanoparticles would enhance the association of HVJ-E with the cell membrane with a magnetic force. However, maghemite particles surface-coated with heparin, but not protamine sulfate, enhanced the transfection efficiency in the analysis of direct injection into the mouse liver in an in vivo model. The size and surface chemistry of magnetic particles could be tailored accordingly to meet specific demands of physical and biological characteristics. Overall, magnetic nanoparticles with different surface modifications can enhance HVJ-E-based gene transfer by modification of the size or charge, which could potentially help to overcome fundamental limitations to gene therapy in vivo.     

基于CRISPR-Cas系统的微生物基因编辑与调控技术

CRISPR-Cas系统的基因编辑与调控技术为原核和真核微生物的遗传改造提供了高效且精确的工具,显著提升了工业底盘细胞的生产能力和多样性。在原核微生物中,大肠杆菌、枯草芽孢杆菌、谷氨酸棒杆菌等工业重要菌株通过CRISPR-Cas系统实现了代谢网络的重构和多基因位点的精确编辑,显著优化了细胞工厂的生产性能。在真核微生物中,酿酒酵母、毕赤酵母、解脂耶氏酵母等模式生物也得益于CRISPR-Cas系统的发展,尤其是CRISPR-Cas9、CRISPR-Cas12a和碱基编辑工具等在基因组精确编辑和调控中的应用,实现了代谢途径的高效优化。高通量基因组编辑技术,如CHASE、CREATE、iCREATE等,结合了CRISPR-Cas系统的强大工具,能够同时对多个基因进行编辑和调控,加速了对复杂表型的优化和代谢网络的解析。本文总结了CRISPR-Cas系统在原核、真核微生物中的应用及其高通量基因组编辑技术的发展,展望了这些技术在工业微生物细胞工厂构建中的潜在应用。     

金沙江下游外来入侵植物入侵风险评估——以乌东德和白鹤滩电站库区为例

【目的】基于金沙江下游外来物种入侵防控需要,以其干流上乌东德和白鹤滩电站库区为例,调查库区内的外来入侵植物并评估入侵风险,为该区域外来植物入侵防控和生态安全建设提供数据支撑。【方法】运用样线、样方调查法结合文献资料掌握库区内外来入侵植物种类组成和入侵现状,采用层次分析法建立外来入侵植物风险评估体系,对其入侵过程、入侵现状以及危害与防治等方面进行综合评估,并根据等级划分标准确定入侵风险等级。【结果】乌东德和白鹤滩电站库区共记录到外来入侵植物103种,隶属28科70属。其中,Ⅰ级风险的外来入侵植物5种,Ⅱ级风险12种,Ⅲ级风险24种,Ⅳ级风险28种,Ⅴ级风险34种。【结论】乌东德和白鹤滩电站库区的外来入侵植物种类较多,但多数处于低风险等级(Ⅳ、Ⅴ级),危害较小,高风险等级(I、Ⅱ级)外来入侵植物共17种。建议重点防控高风险等级的外来入侵植物,尽可能降低其对金沙江下游生态安全的威胁。     

玉米rMrh/Mrh双元转座系统体细胞转座特性分析

基因组中转座子的插入和剪切都会使插入位点的序列发生改变,包括基因突变,进而导致表型变异或基因组进化。Mrh/rMrh是玉米Mutator超家族中首个经遗传学鉴定的双元转座子系统,其中仅有非自主性转座子rMrh完成了基因克隆及序列测定。通过遗传杂交后代的表型分离比确定Mrh活性系中仅有一个自主性转座子Mrh调控报告基因a1位点的rMrh发生转座。为了明确rMrh转座子在玉米体细胞中的转座剪切修复对宿主a1基因功能的影响,以及分处两个遗传位点的自主性Mrh转座子对同一a1位点rMrh剪切转座后序列修复类型的影响,对转座修复位点的PCR扩增产物进行了分子克隆及测序。结果显示,rMrh在玉米体细胞中a1位点转座后产生两种足迹类型,一种是精确修复,另一种是末端反向重复序列（terminal inverted repeat,TIR）残留。同时,在不同遗传位点的自主性转座子Mrh的调控下,rMrh在玉米体细胞中原初位点发生转座剪切时的修复类型相对单一,既能通过精确修复恢复宿主基因功能,也会因为残留碱基导致宿主基因功能的突变,但是不同遗传位点的自主性转座子Mrh的修复产物序列不尽相同。研究结果明确了经典Mrh/rMrh双元转座子系统中rMrh的转座剪切修复特性,在丰富对Mutator转座子遗传特性认识的同时,也为进一步应用这一转座子系统创制玉米新种质资源和功能基因组学遗传材料奠定了理论基础。