Enhancement of bioconversion gangliosides to monosialotetrahexosylganglioside by in situ sialic acid removal and recovery

Monosialotetrahexosylganglioside (GM1) production via bioconversion from gangliosides is promising for industrial application because it has the advantages of a high GM1 yield and an environmentally friendly process. Sialidase hydrolyzes gangliosides to GM1 producing sialic acid as a by‐product, which inhibits the sialidase activity, while the incomplete conversion of gangliosides was indicated by thin‐layer chromatography (TLC) in the presence of sialic acid. The sialic acid showed competitive inhibition on the sialidase activity with an inhibition constant of 0.75 mmol/L. By harnessing the in situ product removal (ISPR) technique, 50 g/L of crude gangliosides was completely converted to GM1 after a 12 h conversion. The GM1 concentration increased from 0.42 to 10.88 g/L in the ISPR system, which was 59.1 % higher than that of the control (6.84 g/L GM1). In addition, sialic acid was recovered simultaneously with a yield of 74.7 %. In summary, the ISPR system improved the bioconversion from gangliosides to GM1 and recovered sialic acid within a one‐step bioprocess.     

一种制备唾液酸磁性表面分子印迹聚合物的新方法

一锅法合成氨基化磁性纳米颗粒Fe₃O₄@NH₂,与对甲酰基苯硼酸(FPBA)反应嫁接硼酸官能团,通过硼酸基与模板唾液酸Neu5Ac分子上的顺式二醇共价反应,将Neu5Ac定向固定于磁性纳米颗粒。以多巴胺(DA)及3-氨基苯硼酸(3-APBA)为功能单体,自聚合反应形成共聚壳层包覆在磁性纳米颗粒的表面,制备得Neu5Ac磁性分子印迹聚合物(Neu5Ac-MMIPs)。通过透射电镜、红外光谱对其形态及结构进行表征,并评价其吸附性能。结果表明,Neu5Ac磁性分子印迹聚合物对Neu5Ac具有较好的吸附量、印迹效率、特异性等优点。通过对Neu5Ac至少5次吸附-洗脱的循环实验表明,MMIPs具有较好的重复再利用能力。     

Assaying sialyltransferase activity with surface plasmon resonance

Here, we describe an activity assay for sialyltransferases based on surface plasmon resonance (SPR). Different natural and synthetic oligosaccharides serving as acceptor substrates for the sialyltransferase ST3Gal-III (EC 2.4.99.6) were immobilized or synthesized on SPR chips. The chip was then exposed to different concentrations of a reaction mixture of ST3Gal-III and CMP-Neu5Ac either by injection or by external application of the reaction mixture to the chip surface. The binding of two lectins, one that specifically recognizes the unmodified acceptor, the other the sialylated oligosaccharide, was utilized to determine the extent of enzymatic turnover. In order to obtain enzymatic activities, the SPR data were correlated to data obtained from a classical radio assay. After regeneration, that is, cleavage of the sialic acid residues by using a sialidase, the chip is available for new experiments. The technique allows the rapid determination of sialyltransferase activity with only nanomolar quantities of acceptor substrates and should be of particular value in cases in which a large variety of samples, including cell lysates, have to be screened for their enzymatic activities.     

Efficient Whole‐Cell Biocatalytic Synthesis of N‐Acetyl‐D‐neuraminic Acid

N‐Acetyl‐D ‐neuraminic acid (Neu5Ac) was efficiently synthesized from lactate and a mixture of N‐acetyl‐D ‐glucosamine (GlcNAc) and N‐acetyl‐D ‐mannosamine (ManNAc) by whole cells. The biotransformation utilized Escherichia coli cells (Neu5Ac aldolase), Pseudomonas stutzeri cells (lactate oxidase components), GlcNAc/ManNAc and lactate. By this process, 18.32±0.56 g/liter Neu5Ac were obtained from 65.61±2.70 g/liter lactate as an initial substrate input. Neu5Ac (98.4±0.4 % purity, 80.87±0.79 % recovery yield) was purified by anionic exchange chromatography. Our results demonstrate that the reported Neu5Ac biosynthetic process can compare favorably with natural product extraction or chemical synthesis processes.     

离子液体1-乙基-3-甲基咪唑醋酸盐对酿酒酵母AY93161的毒性及其酒精发酵过程的影响

为了考察离子液体在由木质纤维原料制备可发酵糖中的残留对后续酒精发酵过程的影响,对离子液体1-乙基-3-甲基咪唑醋酸盐([Emim]Ac)对酿酒酵母AY93161的毒性及其酒精发酵过程的影响进行研究。通过亚甲基蓝染色,利用OLYMPUS CX41显微镜观察不同[Emim]Ac浓度下对数生长期酵母细胞的形态结构,出芽情况及代谢活性,发现在[Emim]Ac浓度高于5 g·L^-1时,酵母细胞的形态结构会发生变化,在[Emim]Ac浓度高于0.1 g·L^-1时,随着[Emim]Ac浓度的增加,酵母的出芽速率及代谢活性降低。通过平板培养和液体悬浮培养测得[Emim]Ac对酵母的半有效浓度EC50和半抑制浓度IC50分别为4.45、7.70 g·L^-1。通过测定不同[Emim]Ac浓度下酵母酒精发酵的过程数据,发现在[Emim]Ac浓度低于0.1 g·L^-1时,对酵母酒精发酵过程几乎无影响,在[Emim]Ac浓度高于0.1 g·L^-1时,对酵母酒精发酵有抑制作用,[Emim]Ac对酵母酒精发酵的抑制作用主要是由其对菌体生长的抑制所致。     

Kinetics of Americium(III) Extraction and Back Extraction with BTP

Abstract

2,6‐Di(5,6‐dipropyl‐1,2,4‐triazin‐3‐yl)pyridine (BTP) extracts trivalent actinides from nitric acid with high separation factors over the lanthanides. The kinetics of americium(III) extraction and back extraction of this extraction system was studied in a constant‐interface stirred cell. The americium(III) extraction rate was found to be independent of the stirring speed. This means that the rate of mass transfer is limited by a slow chemical complexation reaction (“chemical regime”). The americium(III) extraction rate was found to increase linearly with BTP concentration. Nitric acid concentration had a strong influence on the rate of the americium(III) extraction, due to its influence on the free extractant concentration. The addition of ammonium nitrate did not affect the rate of americium(III) extraction. By investigating the influence of the interfacial area on the americium(III) extraction rate, the interface was identified as the site of the chemical reaction. The americium(III) back extraction rate increased linearly with the stirring speed, indicating that the back extraction is limited by diffusion. The extraction and the back extraction rates could be calculated by a simple model based on equilibrium data for the co‐extraction of americium(III) and nitric acid.     

Effects of the Selective Serotonin Reuptake Inhibitor Fluoxetine on Developing Neural Circuits in a Model of the Human Fetal Cortex

The developing prenatal brain is particularly susceptible to environmental disturbances. During prenatal brain development, synapses form between neurons, resulting in neural circuits that support complex cognitive functions. In utero exposure to environmental factors such as pharmaceuticals that alter the process of synapse formation increases the risk of neurodevelopmental abnormalities. However, there is a lack of research into how specific environmental factors directly impact the developing neural circuitry of the human brain. For example, selective serotonin reuptake inhibitors are commonly used throughout pregnancy to treat depression, yet their impact on the developing fetal brain remains unclear. Recently, human brain models have provided unprecedented access to the critical window of prenatal brain development. In the present study, we used human neurons and cortical spheroids to determine whether the selective serotonin reuptake inhibitor fluoxetine alters neurite and synapse formation and the development of spontaneous activity within neural circuits. We demonstrate that cortical spheroids express serotonin transporter, thus recapitulating the early developmental expression of serotonin transporter associated with cortical pyramidal neurons. Cortical spheroids also appropriately express serotonin receptors, such as synaptic 5-HT2A and glial 5-HT5A. To determine whether fluoxetine can affect developing neural circuits independent of serotonergic innervation from the dorsal and medial raphe nuclei, we treated cortical neurons and spheroids with fluoxetine. Fluoxetine alters neurite formation in a dose-dependent fashion. Intriguingly, in cortical spheroids, neither acute nor chronic fluoxetine significantly altered excitatory synapse formation. However, only acute, but not chronic fluoxetine exposure altered inhibitory synaptogenesis. Finally, fluoxetine reversibly suppresses neuronal activity in a dose-dependent manner. These results demonstrate that fluoxetine can acutely alter synaptic function in developing neural circuits, but the effects were not long-lasting. This work provides a foundation for future studies to combine serotonergic innervation with cortical spheroids and assess the contributions of fluoxetine-induced alterations in serotonin levels to brain development.     

N-Acetylneuraminic Acid Production in Escherichia coli Lacking N-Acetylglucosamine Catabolic Machinery

N-Acetylneuraminic acid (Neu5Ac, also referred to as sialic acid) is a nine-carbon sugar found on cell surfaces in higher animals. With key roles in inflammation, brain development, viral adhesion, and production of therapeutic glycoproteins, access to Neu5Ac is essential. We demonstrate that disruption of the N-acetylglucosamine (GlcNAc) degradation pathway by deletion of nagA and bypassing the GlmMU pathway for uridine diphosphate-GlcNAc production by expression of the Saccharomyces cerevisiae genes agm1 and uap1 improves the NeuCB-based direct cell culture approach to Neu5Ac production. The Escherichia coli strain BRL04 (nanT^?, nanA^?, and nagA^?) transformed with a polycistronic inducible expression vector encoding Agm1, Uap1, NeuB, and NeuC, cultivated in a shake-flask and fed with glycerol and GlcNAc produced Neu5Ac at 3.7 g/L (87% conversion from GlcNAc). At the 2 L bioreactor scale, production reached 7.3 g/L at a reduced conversion of 52%. These promising results suggest that this production strain is capable of generating Neu5Ac via high-density cultivation; it remains to be seen if careful control of GlcNAc feeding rate can be optimized to maximize yield.     

Quantitative Standards of 4-O-Acetyl- and 9-O-Acetyl-N-Acetylneuraminic Acid for the Analysis of Plasma and Serum

N-Acetylneuraminic acid (sialic acid, Neu5Ac) is one of a large, diverse family of nine-carbon monosaccharides that play roles in many biological functions such as immune response. Neu5Ac has previously been identified as a potential biomarker for the presence and pathogenesis of cardiovascular disease (CVD), diabetes and cancer. More recent research has highlighted acetylated sialic acid derivatives, specifically Neu5,9Ac₂, as biomarkers for oral and breast cancers, but advances in analysis have been hampered due to a lack of commercially available quantitative standards. We report here the synthesis of 9-O- and 4-O-acetylated sialic acids (Neu5,9Ac₂ and Neu4,5Ac₂) with optimisation of previously reported synthetic routes. Neu5,9Ac₂ was synthesised in 1 step in 68 % yield. Neu4,5Ac₂ was synthesised in 4 steps in 39 % overall yield. Synthesis was followed by analysis of these standards via quantitative NMR (qNMR) spectroscopy. Their utilisation for the identification and quantification of specific acetylated sialic acid derivatives in biological samples is also demonstrated.     

Evaluation of Class IIa Histone Deacetylases Expression and In Vivo Epigenetic Imaging in a Transgenic Mouse Model of Alzheimer’s Disease

Epigenetic regulation by histone deacetylase (HDAC) is associated with synaptic plasticity and memory formation, and its aberrant expression has been linked to cognitive disorders, including Alzheimer’s disease (AD). This study aimed to investigate the role of class IIa HDAC expression in AD and monitor it in vivo using a novel radiotracer, 6-(tri-fluoroacetamido)-1-hexanoicanilide ([¹⁸F]TFAHA). A human neural cell culture model with familial AD (FAD) mutations was established and used for in vitro assays. Positron emission tomography (PET) imaging with [¹⁸F]TFAHA was performed in a 3xTg AD mouse model for in vivo evaluation. The results showed a significant increase in HDAC4 expression in response to amyloid-β (Aβ) deposition in the cell model. Moreover, treatment with an HDAC4 selective inhibitor significantly upregulated the expression of neuronal memory-/synaptic plasticity-related genes. In [¹⁸F]TFAHA-PET imaging, whole brain or regional uptake was significantly higher in 3xTg AD mice compared with WT mice at 8 and 11 months of age. Our study demonstrated a correlation between class IIa HDACs and Aβs, the therapeutic benefit of a selective inhibitor, and the potential of using [¹⁸F]TFAHA as an epigenetic radiotracer for AD, which might facilitate the development of AD-related neuroimaging approaches and therapies.     

Structure-guided saturation mutagenesis of N-acetylneuraminic acid lyase for the synthesis of sialic acid mimetics

Analogues of N-acetylneuraminic acid (sialic acid, NANA, Neu5Ac), including 6-dipropylcarboxamides, have been found to be selective and potent inhibitors of influenza sialidases. Sialic acid analogues are, however, difficult to synthesize by traditional chemical methods and the enzyme N-acetylneuraminic acid lyase (NAL) has previously been used for the synthesis of a number of analogues. The activity of this enzyme towards 6-dipropylcarboxamides is, however, low. Here, we used structure-guided saturation mutagenesis to produce variants of NAL with improved activity and specificity towards 6-dipropylcarboxamides. Three residues were targeted for mutagenesis, Asp191, Glu192 and Ser208. Only substitution at position 192 produced significant improvements in activity towards the dipropylamide. One variant, E192N, showed a 49-fold improvement in catalytic efficiency towards the target analogue and a 690-fold shift in specificity from sialic acid towards the analogue. These engineering efforts provide a scaffold for the further tailoring of NAL for the synthesis of sialic acid mimetics.     

An Extracellular Perspective on CNS Maturation: Perineuronal Nets and the Control of Plasticity

During restricted time windows of postnatal life, called critical periods, neural circuits are highly plastic and are shaped by environmental stimuli. In several mammalian brain areas, from the cerebral cortex to the hippocampus and amygdala, the closure of the critical period is dependent on the formation of perineuronal nets. Perineuronal nets are a condensed form of an extracellular matrix, which surrounds the soma and proximal dendrites of subsets of neurons, enwrapping synaptic terminals. Experimentally disrupting perineuronal nets in adult animals induces the reactivation of critical period plasticity, pointing to a role of the perineuronal net as a molecular brake on plasticity as the critical period closes. Interestingly, in the adult brain, the expression of perineuronal nets is remarkably dynamic, changing its plasticity-associated conditions, including memory processes. In this review, we aimed to address how perineuronal nets contribute to the maturation of brain circuits and the regulation of adult brain plasticity and memory processes in physiological and pathological conditions.     

Sex pheromone components of female smaller tea tortrix moth,Adoxophyes sp. (Lepidoptera: Tortricidae) in Taiwan

(Z)-11-Teiradecenyl acetate (Z11-14: Ac) and (Z)-9-tetrade-cenyl acetate (Z9–14: Ac) were isolated as major sex pheromone components from the female tips of the smaller tea tortrix moth,Adoxophyes sp., in Taiwan. The average amount ofZ11- andZ9–14: Ac in each female gland was 24.9 and 14.1 ng, in a ratio of 6436, respectively. When compared to a closely related Japanese strain, which used theZ9-14: Ac,Z11-14 Ac,E11–14:Ac, 10-Me-12:Ac four-component system (in a ratio of 633142), the Taiwan formulation of two components caught significantly more Taiwan males than the Japanese formulation of four components.     

Ceftriaxone Treatment Weakens Long-Term Synaptic Potentiation in the Hippocampus of Young Rats

Disrupted glutamate clearance in the synaptic cleft leads to synaptic dysfunction and neurological diseases. Decreased glutamate removal from the synaptic cleft is known to cause excitotoxicity. Data on the physiological effects of increased glutamate clearance are contradictory. This study investigated the consequences of ceftriaxone (CTX), an enhancer of glutamate transporter 1 expression, treatment on long-term synaptic potentiation (LTP) in the hippocampus of young rats. In this study, 5-day administration of CTX (200 mg/kg) significantly weakened LTP in CA3-CA1 synapses. As shown by electrophysiological recordings, LTP attenuation was associated with weakening of N-Methyl-D-aspartate receptor (NMDAR)-dependent signaling in synapses. However, PCR analysis did not show downregulation of NMDAR subunits or changes in the expression of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) subunits. We assume that extracellular burst stimulation activates fewer synapses in CTX-treated animals because increased glutamate reuptake results in reduced spillover, and neighboring synapses do not participate in neurotransmission. Attenuation of LTP was not accompanied by noticeable behavioral changes in the CTX group, with no behavioral abnormalities observed in the open field test or Morris water maze test. Thus, our experiments show that increased glutamate clearance can impair long-term synaptic plasticity and that this phenomenon can be considered a potential side effect of CTX treatment.     

Development of New and Selective Trypanosoma cruzi trans‐Sialidase Inhibitors from Sulfonamide Chalcones and Their Derivatives

A series of sulfonamide‐containing hydroxylated chalcone ( 4 – 7 ) and quinolinone ( 8 , 9 ) derivatives was synthesised and tested for inhibition of the trans‐sialidase from Trypanosoma cruzi (TcTS). IC₅₀ values for these inhibitors ranged from 0.6 to 7.3 μM , with the dihydroxylated (catechol) derivatives being the tightest binders. Full kinetic analyses of inhibition were performed for these catechol derivatives, both for the transglycosylation reaction in the presence of lactose and for the hydrolysis reaction in its absence. Competitive inhibition was seen in each case with K_i values for 5 , 7 and 9 of 2.0, 2.2 and 0.2 μM , respectively, in the absence of lactose, and 4.6, 3.7 and 0.4 μM in its presence. None of the compounds tested showed any significant inhibition of the human sialidase Neu2, at concentrations up to 200 μM .     

Somatostatin,a Presynaptic Modulator of Glutamatergic Signal in the Central Nervous System

Somatostatin is widely diffused in the central nervous system, where it participates to control the efficiency of synaptic transmission. This peptide mainly colocalizes with GABA, in inhibitory, GABA-containing interneurons from which it is actively released in a Ca²⁺ dependent manner upon application of depolarizing stimuli. Once released in the synaptic cleft, somatostatin acts locally, or it diffuses in the extracellular space through “volume diffusion”, a mechanism(s) of distribution which mainly operates in the cerebrospinal fluid and that assures the progression of neuronal signalling from signal-secreting sender structures towards receptor-expressing targeted neurons located extrasynaptically, in a non-synaptic, inter-neuronal form of communication. Somatostatin controls the efficiency of central glutamate transmission by either modulating presynaptically the glutamate exocytosis or by metamodulating the activity of glutamate receptors colocalized and functionally coupled with somatostatin receptors in selected subpopulations of nerve terminals. Deciphering the role of somatostatin in the mechanisms of “volume diffusion” and in the “receptor-receptor interaction” unveils new perspectives in the central role of this fine tuner of synaptic strength, paving the road to new therapeutic approaches for the cure of central disorders.     

小鼠激活素受体相互作用蛋白5的基因克隆

目的克隆新的小鼠激活素受体相互作用蛋白5(ActRIP5)基因。方法应用酵母双杂交技术,筛选出与激活素ⅡA型受体(ActRⅡA)相互作用蛋白的基因片段,再以此基因片段作为探针,从小鼠脑cDNA文库中钓取ActRIP5cDNA。采用哺乳动物细胞双杂交系统,进一步确定ActRIP5与ActRⅡA的相互作用。采用RT-PCR检测ActRIP5mRNA在组织中的转录。结果克隆的ActRIP5全编码基因长1839bp,编码145个氨基酸残基,与ActRⅡA具有特异结合作用。ActRIP5mR-NA在小鼠多种组织中均可检出。结论ActRIP5属于ActRIP家族新成员,可以与ActRⅡA相互作用。     

Emerging Roles of Astrocyte Kir4.1 Channels in the Pathogenesis and Treatment of Brain Diseases

Inwardly rectifying Kir4.1 channels in astrocytes mediate spatial potassium (K⁺) buffering, a clearance mechanism for excessive extracellular K⁺, in tripartite synapses. In addition to K⁺ homeostasis, astrocytic Kir4.1 channels also play an essential role in regulating extracellular glutamate levels via coupling with glutamate transporters. Moreover, Kir4.1 channels act as novel modulators of the expression of brain-derived neurotrophic factor (BDNF) in astrocytes. Specifically, inhibition of astrocytic Kir4.1 channels elevates extracellular K⁺ and glutamate levels at synapses and facilitates BDNF expression in astrocytes. These changes elevate neural excitability, which may facilitate synaptic plasticity and connectivity. In this article, we summarize the functions and pharmacological features of Kir4.1 channels in astrocytes and highlight the importance of these channels in the treatment of brain diseases. Although further validation in animal models and human patients is required, astrocytic Kir4.1 channel could potentially serve as a novel therapeutic target for the treatment of depressive disorders and epilepsy.     

The chemistry and biology of trypanosomal trans-sialidases: virulence factors in Chagas disease and sleeping sickness

trans-Sialidases constitute a special group of the sialidase family. They occur in some trypanosome species and, in a unique reversible reaction, transfer sialic acids from one glycosidic linkage with galactose (donor) to another galactose (acceptor), to form (α2-3)-sialyl linkages. Trypanosomes cause such devastating human diseases as Chagas disease in South America (Trypanosoma cruzi) or sleeping sickness in Africa (Trypanosoma brucei). The trans-sialidases strongly contribute to the pathogenicity of the trypanosomes by scavenging sialic acids from the host or blood meal to coat the parasite surface; this aids their survival strategy in the insect's intestine, and in the blood circulation or cells of the host, and serves to compromise the immune system of the human or animal host. American and African trypanosomes express trans-sialidases at different stages of their vector/host development. They are transmitted to humans by insect vectors (tsetse fly or other insect "bug" species). trans-Sialidase activity with varying linkage specificity has also been found in a few bacteria species and in human serum. trans-Sialidases are of increasing practical importance for the chemo-enzymatic synthesis of sialylated glycans. The search for appropriate inhibitors of trans-sialidases and vaccination strategies is intensifying, as less toxic medicaments for the treatment of these widespread and often chronic tropical diseases are required.