Exchange of domains of glutamate dehydrogenase from the hyperthermophilic archaeon Pyrococcus furiosus and the mesophilic bacterium Clostridium difficile: effects on catalysis, thermoactivity and stability

The glutamate dehydrogenase gene from the hyperthermophilicarchaeon Pyrococcus furiosus has been functionally expressedin Escherichia coli under the control of the X, PL promoter.The P.furiosus glutamate dehydrogenase amounted to 20% of thetotal E.coli cell protein, and the vast majority consisted ofhexamers. Following activation by heat treatment, an enzymecould be purified from E.coli that was indistinguishable fromthe glutamate dehydrogenase purified from P.furiosus. Hybridgenes, that consisted of the coding regions for the homologousglutamate dehydrogenases from P.furiosus and the mesophilicbacterium Clostridium difficile, were constructed and successfullyexpressed in E.coli. One of the resulting hybrid proteins, containingthe glutamate binding domain of the C.difficile enzyme and thecofactor binding domain of the P.furiosus enzyme, did not showa detectable activity. In contrast, the complementary hybridcontaining the P.furiosus glutamate and the C.difficile cofactorbinding domain was a catalytically active hexamer that showeda reduced substrate affinity but maintained efficient cofactorbinding with the specificity found in the Clostridium symbiosumenzyme. Compared with the C.difficile glutamate dehydrogenase,the archaeal-bacterial hybrid is slightly more thermoactive,less thermostable but much more stable towards guanidinium chloride-inducedinactivation and denaturation     

A New Player in the Hippocampus: A Review on VGLUT3+ Neurons and Their Role in the Regulation of Hippocampal Activity and Behaviour

Glutamate is the most abundant excitatory amino acid in the central nervous system. Neurons using glutamate as a neurotransmitter can be characterised by vesicular glutamate transporters (VGLUTs). Among the three subtypes, VGLUT3 is unique, co-localising with other “classical” neurotransmitters, such as the inhibitory GABA. Glutamate, manipulated by VGLUT3, can modulate the packaging as well as the release of other neurotransmitters and serve as a retrograde signal through its release from the somata and dendrites. Its contribution to sensory processes (including seeing, hearing, and mechanosensation) is well characterised. However, its involvement in learning and memory can only be assumed based on its prominent hippocampal presence. Although VGLUT3-expressing neurons are detectable in the hippocampus, most of the hippocampal VGLUT3 positivity can be found on nerve terminals, presumably coming from the median raphe. This hippocampal glutamatergic network plays a pivotal role in several important processes (e.g., learning and memory, emotions, epilepsy, cardiovascular regulation). Indirect information from anatomical studies and KO mice strains suggests the contribution of local VGLUT3-positive hippocampal neurons as well as afferentations in these events. However, further studies making use of more specific tools (e.g., Cre-mice, opto- and chemogenetics) are needed to confirm these assumptions.     

油酰谷氨酸的合成

通过油酸和三氯化磷反应制备了油酰氯,再用油酰氯与谷氨酸单钠盐反应制备了目标产物油酰-L-谷氨酸。并讨论了原料配比、反应温度和反应时间等因素对氯化反应的影响,确定最佳氯化条件为:反应温度70~80℃,n(三氯化磷)∶n(油酸)=0.3~0.5∶1,反应时间4~6h。同时还探讨了反应温度、物料配比、pH值和盐酸用量对产物油酰-L-谷氨酸产率的影响,确定最佳制备条件为:n(油酰氯)∶n(谷氨酸钠)=1.0~1.1∶1,反应温度为0~10℃,反应过程和酸化结束时的pH分别为11~13和0.5~1。     

味精闭环生产新工艺

介绍通过膜分离技术开发出来的味精闭环生产工艺路线。该工艺采用无机陶瓷超滤膜将发酵液超滤;用浓硫酸水解截留液;过滤水解液,在滤液中得水解产物;混合渗透液与水解过滤液,进行谷氨酸结晶;最后用味精结晶母液纯化谷氨酸结晶。该工艺采用蒸发与冷却耦合连续结晶工艺,结晶收率高,且节能效果显著。     

Comprehensive Research on Past and Future Therapeutic Strategies Devoted to Treatment of Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a rapidly debilitating fatal neurodegenerative disorder, causing muscle atrophy and weakness, which leads to paralysis and eventual death. ALS has a multifaceted nature affected by many pathological mechanisms, including oxidative stress (also via protein aggregation), mitochondrial dysfunction, glutamate-induced excitotoxicity, apoptosis, neuroinflammation, axonal degeneration, skeletal muscle deterioration and viruses. This complexity is a major obstacle in defeating ALS. At present, riluzole and edaravone are the only drugs that have passed clinical trials for the treatment of ALS, notwithstanding that they showed modest benefits in a limited population of ALS. A dextromethorphan hydrobromide and quinidine sulfate combination was also approved to treat pseudobulbar affect (PBA) in the course of ALS. Globally, there is a struggle to prevent or alleviate the symptoms of this neurodegenerative disease, including implementation of antisense oligonucleotides (ASOs), induced pluripotent stem cells (iPSCs), CRISPR-9/Cas technique, non-invasive brain stimulation (NIBS) or ALS-on-a-chip technology. Additionally, researchers have synthesized and screened new compounds to be effective in ALS beyond the drug repurposing strategy. Despite all these efforts, ALS treatment is largely limited to palliative care, and there is a strong need for new therapeutics to be developed. This review focuses on and discusses which therapeutic strategies have been followed so far and what can be done in the future for the treatment of ALS.     

Neuroinflammation in Schizophrenia: The Key Role of the WNT/β-Catenin Pathway

Schizophrenia is a very complex syndrome involving widespread brain multi-dysconnectivity. Schizophrenia is marked by cognitive, behavioral, and emotional dysregulations. Recent studies suggest that inflammation in the central nervous system (CNS) and immune dysfunction could have a role in the pathogenesis of schizophrenia. This hypothesis is supported by immunogenetic evidence, and a higher incidence rate of autoimmune diseases in patients with schizophrenia. The dysregulation of the WNT/β-catenin pathway is associated with the involvement of neuroinflammation in schizophrenia. Several studies have shown that there is a vicious and positive interplay operating between neuroinflammation and oxidative stress. This interplay is modulated by WNT/β-catenin, which interacts with the NF-kB pathway; inflammatory factors (including IL-6, IL-8, TNF-α); factors of oxidative stress such as glutamate; and dopamine. Neuroinflammation is associated with increased levels of PPARγ. In schizophrenia, the expression of PPAR-γ is increased, whereas the WNT/β-catenin pathway and PPARα are downregulated. This suggests that a metabolic-inflammatory imbalance occurs in this disorder. Thus, this research’s triptych could be a novel therapeutic approach to counteract both neuroinflammation and oxidative stress in schizophrenia.     

Effect of glutamate and inosinate on sensory and instrumental texture of extruded products

Expanded products have been developed by extrusion of non-conventional highly nutritious raw materials such as amaranth and chickpea blended with bovine lung. As sensory acceptance of these snacks is restricted, this study aimed at improving their texture, through the addition of monosodium glutamate (MSG) and disodium inosinate (IMP) flavor enhancers to the feeding material, or to the flavor added after the extrusion. Sensory and mechanical analyses showed that both enhancers affected texture, assessed by sensory and instrumental methods. Addition of IMP together with MSG to the chickpea-based snacks presented the best results. This beneficial effect was not observed in the amaranth-based snack, suggesting that IMP and MSG can favorably impact texture of extruded products depending on the amount and type of protein present.     

超声对谷氨酸棒杆菌发酵L-异亮氨酸的影响

为解决谷氨酸棒杆菌发酵产异亮氨酸适应期较长,菌体细胞膜通透性差,异亮氨酸分泌速率慢的问题,实验通过在发酵罐内安装超声棒,探究超声对谷氨酸棒杆菌整个发酵过程中生物量及产酸的影响。研究从超声周期、超声功率、超声频率、超声时间和超声模式5个方面,探究了菌体生物量及产酸的最优条件。结果表明,使用80 W/L、18 kHz的超声波,在菌体适应期、对数生长期及平稳期分别超声2、6、1 h,超声模式设置为开10 s、停30 s,菌体发酵适应阶段缩短至2 h以内,菌体快速进入对数生长期,且对数生长期从2 h延续到24 h,直至40 h结束菌体活力依旧很强,最终菌体干重达到了41.0 g/L,比未超声提高了74.5%; L-异亮氨酸产量达到了39.0g/L,比未超声产酸量提升了69.6%。超声产生的微扰动有利于细胞增殖,同时产生的机械剪切作用增加了细胞膜的通透性,提高了产酸能力。     

接种颗粒污泥UASB反应器处理味精-卡那霉素混合废水

采用屠宰废水中培养的颗粒污泥接种启动中温（３５±１）℃ ＵＡＳＢ反应器处理味精－卡那霉素混合废水,反应器能随较高浓度的硫酸盐,氨氮和的氯化物。当ＨＲＴ为２－３ｈ,容积负荷率可达３５－４０ｋｇ ＣＯＤ／（ｍ＾３．ｄ）,ＣＯＤ去除率为７５％－８０％,进水ＣＯＤ／ＳＯ＾２－４可低至４－５。     

Stereocontrolled Synthesis and Pharmacological Evaluation of Azetidine‐2,3‐Dicarboxylic Acids at NMDA Receptors

The four stereoisomers of azetidine‐2,3‐dicaroxylic acid (L ‐trans‐ADC, L ‐cis‐ADC, D ‐trans‐ADC, and D ‐cis‐ADC) were synthesized in a stereocontrolled fashion following two distinct strategies: one providing the two cis‐ADC enantiomers and one giving access to the two trans‐ADC enantiomers. The four azetidinic amino acids were characterized in a radioligand binding assay ([³H]CGP39653) at native NMDA receptors: L ‐trans‐ADC showed the highest affinity (K_i=10 μM ) followed by the D ‐cis‐ADC stereoisomer (21 μM ). In contrast, the two analogues L ‐cis‐ADC and D ‐trans‐ADC were low‐affinity ligands (>100 and 90 μM , respectively). Electrophysiological characterization of the ADC compounds at the four NMDA receptor subtypes NR1/NR2A, NR1/NR2B, NR1/NR2C, and NR1/NR2D expressed in Xenopus oocytes showed that L ‐trans‐ADC displayed the highest agonist potency at NR1/NR2D (EC₅₀=50 μM ), which was 9.4‐, 3.4‐, and 1.9‐fold higher than the respective potencies at NR1/NR2A–C. D ‐cis‐ADC was shown to be a partial agonist at NR1/NR2C and NR1/NR2D with medium‐range micromolar potencies (EC₅₀=720 and 230 μM , respectively). A subsequent in silico ligand–protein docking study suggested an unusual binding mode for these amino acids in the agonist binding site.