Efficient biosynthesis of γ-aminobutyric acid by rationally engineering the catalytic pH range of a glutamate decarboxylase from Lactobacillus plantarum / 生物工程学报

γ-aminobutyric acid can be produced by a one-step enzymatic reaction catalyzed by glutamic acid decarboxylase. The reaction system is simple and environmentally friendly. However, the majority of GAD enzymes catalyze the reaction under acidic pH at a relatively narrow range. Thus, inorganic salts are usually needed to maintain the optimal catalytic environment, which adds additional components to the reaction system. In addition, the pH of solution will gradually rise along with the production of γ-aminobutyric acid, which is not conducive for GAD to function continuously. In this study, we cloned the glutamate decarboxylase LpGAD from a Lactobacillus plantarum capable of efficiently producing γ-aminobutyric acid, and rationally engineered the catalytic pH range of LpGAD based on surface charge. A triple point mutant LpGADS24R/D88R/Y309K was obtained from different combinations of 9 point mutations. The enzyme activity at pH 6.0 was 1.68 times of that of the wild type, suggesting the catalytic pH range of the mutant was widened, and the possible mechanism underpinning this increase was discussed through kinetic simulation. Furthermore, we overexpressed the Lpgad and LpgadS24R/D88R/Y309K genes in Corynebacterium glutamicum E01 and optimized the transformation conditions. An optimized whole cell transformation process was conducted under 40 ℃, cell mass (OD600) 20, 100 g/L l-glutamic acid substrate and 100 μmol/L pyridoxal 5-phosphate. The γ-aminobutyric acid titer of the recombinant strain reached 402.8 g/L in a fed-batch reaction carried out in a 5 L fermenter without adjusting pH, which was 1.63 times higher than that of the control. This study expanded the catalytic pH range of and increased the enzyme activity of LpGAD. The improved production efficiency of γ-aminobutyric acid may facilitate its large-scale production.

Effect of sleep fragmentation on postoperative cognitive dysfunction and hippocampal glutamatergic metabolism in aged mice anesthetized with isoflurane / 中华麻醉学杂志

Objective:To evaluate the effect of sleep fragmentation on postoperative cognitive dysfunction (POCD) and hippocampal glutaminergic metabolism in aged mice anesthetized with isoflurane.Methods:Forty healthy SPF-grade male C57BL/6J mice, aged 18 months, weighing 20-30 g, were divided into 4 groups ( n= 10 each) by the random number table method: normal control group (group C), sleep fragmentation group (group SF), isoflurane anesthesia/surgery group (group I/S), and sleep fragmentation plus isoflurane anesthesia/surgery group (group SF+ I/S). Group C did not received any treatment. Group SF received sleep fragmentation for 24 h. The right carotid artery exposure was performed under isoflurane anesthesia in group I/S. Group SF+ I/S received isoflurane anesthesia/right carotid artery exposure at 24 h after sleep fragmentation. The metabolic levels of glutamate (Glu), glutamine (Gln), Glu/Gln complex (Glx), and N-acetylaspartate (NAA) and their ratio to creatine (Cr) were measured by in vivo 9.4T hydrogen proton magnetic resonance spectroscopy at 2 h after anaesthesia. Y maze and Morris water maze tests were used to evaluate the cognitive function at 1-7 days after surgery. The mice were sacrificed after the behavioral testing, brain tissues were immediately obtained, and the number of Nissl bodies and density of dendritic spines in the hippocampal CA1 region were measured by Nissl staining and Golgi staining, respectively. Results:Compared with group C, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr, Gln/Cr and Glx/Cr in the hippocampal CA1 region were increased, and the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in SF, I/S and SF+ I/S groups ( P<0.05). Compared with group SF and group I/S, the percentage of exploration time and shuttle times at the novel arm were significantly decreased, the number of crossing the original platform was decreased, the time of stay at the target quadrant was shortened, the ratios of Glu/Cr and Glx/Cr in hippocampal CA1 region was increased, the ratio of NAA/Cr was decreased, and the number of Nissl bodies and density of dendritic spines were decreased in group SF+ I/S ( P<0.05). Conclusions:Sleep fragmentation exacerbates POCD in aged mice anesthetized with isoflurane, and the mechanism is related to nerve injury induced by abnormality in hippocampal glutaminergic metabolism excitability.

Improvement effects of aucubin on ADHD-like behaviors in mice via the inhibition of excessive astrocytic activation / 中华行为医学与脑科学杂志

Objective:To investigate the effect of aucubin on behaviors and excessive activation of astrocytic in attention deficit/hyperactivity disorder (ADHD) model mice.Methods:Twelve wild-type C57BL/6 pregnant mice (female, clean grade) were intraperitoneally administered with esketamine (15 mg/kg) to establish an ADHD model in offspring mice. The offspring mice were divided into control+ saline group, control+ aucubin group, Ketamine+ saline group and Ketamine+ aucubin group according to the nest matching principle with 15 in each group.At 14 days after birth, mice in the control+ aucubin group and Ketamine+ aucubin group were administered with aucubin (5 mg/kg, once a day) by gavage for 5 days. Mice in control+ saline group and Ketamine+ saline group were administered with equal volume of 0.9% sodium chloride solution. The offspring mice were housed with their mothers in the same cage until 21 days after birth. Twenty-one days after birth, the offspring mice were evaluated by open field test and elevated plus maze tests. Immunofluorescence assay was used to detect the expression of glutamate decarboxylase 2 (GAD2), γ- aminobutyric acid (GABA) and glial fibrillary acidic protein (GFAP) in the amygdala. The morphological changes of astrocytes were quantitatively analyzed by Sholl analysis. GraphPad Prim 9.0.1 software was used for statistical analysis. The comparison of multiple groups was conducted by one-way ANOVA or Kruskal-Wallis test.Results:(1)The results of behavioral experiments showed that the total distance traveled in the open field test and the residence time in open arm of the elevated plus maze were statistically significant ( F=236.90, H=39.92, both P<0.001). The total distance ((7 044±249)mm, (22 891±2 175)mm, P<0.05) and the residence time in open arm(12.69(9.86, 17.24)s, 2.72(0.57, 3.87)s, P<0.05) of mice in Ketamine+ saline group were both higher than those in control+ saline group.The total distance((22 891±2 175)mm, (8 252±839)mm, P<0.05) and the the residence time in open arm(5.45(1.13, 10.99)s, 12.69(9.86, 17.24)s, P<0.05) of Ketamine+ aucubin group were both lower than those of Ketamine+ saline group.(2)The immunofluorescence results showed that the levels of GAD2, GABA and GFAP intensity in amygdala of mice in the four groups were statistically significant ( F=145.50, 50.08, 53.83, all P<0.05). Compared with control+ saline group, the fluorescence intensities of GAD2 ((100.00±9.60)%, (24.86±4.14)%, P<0.05) and GABA ((100.00±16.84))%, (25.48±5.70)%, P<0.05) of Ketamine+ saline group were down-regulated, and the GFAP((100.00±18.02)%, (223.80±25.85)%, P<0.05) was up-regulated. Compared with Ketamine+ saline group, the fluorescence intensities of GAD2 ((24.86±4.14)%, (56.08±6.55)%, P<0.05) and GABA((25.48±5.70)%, (52.59±15.74)%, P<0.05) in Ketamine+ aucubin group were up-regulated, but the fluorescence intensity of GFAP ((223.80±25.85)%, (157.10±22.10)%, P<0.05) was down-regulated.(3)Sholl analysis indicated that the number of the intersections between the astrocyte processes or the branches of astrocyte processes was statistically significant in the 4 groups ( F=12.47, P<0.05). Compared with control+ saline group, the number of the intersections in Ketamine+ saline group((2.07±0.48), (1.67±0.72), P<0.05) increased. While the number of the intersections in Ketamine+ aucubin group was lower than that of Ketamine+ saline group ((1.20±0.78), (2.07±0.48), P<0.05). Conclusion:Aucubin administration can alleviate ADHD-like behaviors in offspring mice, and the mechanism may be associated with the inhibition of excessive astrocytic activation.

Effect of hydroxysafflor yellow a on γ-aminobutyric acid and glutamic acid levels in the inferior colliculus of rats with tinnitus / 国际中医中药杂志

Objective:To evaluate the therapeutic effect of hydroxysafflor yellow A (HYA) on rats with tinnitus and investigate its influence on γ-aminobutyric acid (GABA) and glutamic acid (Glu) levels of inferior colliculus.Methods:The model of rats with tinnitus received an injection of sodium salicylate and "water-drinking suppression" was extablished, and then were divided into four groups with random number table method: normal group, model group, positive control (carbamazepine 5 mg/kg) and HYA (20 mg/kg) groups. Animals were intraperitoneally injected for 15 days. The recovery time of water-drinking suppression of all groups were recorded. The threshold value of auditory brainstem response (ABR) under the different frequency (4, 12, 20 and 28 kHz) in each rat was measured. The levels of GABA and Glu in inferior colliculus in rats with tinnitus were detected by LC-MS/MS.Results:Compared with the model group, the recovery time of water drinking suppression [(3.55±0.69)d vs.(1.83±0.58)d] in HYA group was significantly prolonged ( P<0.01). Compared with the model group, the threshold value of ABR under different frequency (4, 12, 20 and 28 kHz) were significantly reduced in HYA group ( P<0.01). The GABA levels [(2.25±0.26) μmol/g vs.(1.96±0.19)μmol/g] in inferior colliculus of tinnitus rats in HYA group was significantly increased ( P<0.05) while the Glu levels [(2.95±0.34)μmol/g vs.(3.71±0.39)μmol/g] were significantly decreased ( P<0.01). Conclusion:HYA treatment could relieve tinnitus symptoms induced by sodium salicylate, which might be related to the recovery of excitatory/inhibitory neurotransmitter balance.

Research progress in comorbid bipolar disorder and obsessive-compulsive disorder / 中国基层医药

Bipolar disorder and obsessive-compulsive disorder comorbidity is becoming more and more common and has aroused clinical physicians' increasing interest. This review summarizes the epidemiology, clinical characteristics, neurobiochemistry, and treatment of comorbid bipolar disorder and obsessive-compulsive disorder and discusses the neurobiochemical mechanism. Findings from this review will provide evidence for identifying the clinical symptoms of bipolar disorder and obsessive-compulsive disorder comorbidity and for treatment of the comorbidity.

Effects of Naringin on neuronal apoptosis in mice with memory consolidation disorder / 国际中医中药杂志

Objective:To observe the effect of Naringin on neuronal apoptosis in mice with memory consolidation disorderinduced by sodium nitrite.Methods:Fifty mice were randomly divided into blank group, model group, standardized protocol group, high-dose Naringin group and low-dose Naringin group, with 10 mice in each group. The standardized protocol group was given Donepezil 1 mg/kg, the Naringin high and low dose groups were gavaged with Naringin solution 100 and 50 mg/(kg·d), blank group and model group were gavaged with equal volume of distilled water once a day for 21 days. The model was established on the 22nd day. The blank group was intraperitoneally injected with normal saline, and the other groups were intraperitoneally injected with 100 mg/(kg·d) sodium nitrite solution for 7 days. The cognitive ability of mice in each group was evaluated by platform jumping test, and the hippocampal synaptic structure was observed by electron microscope. The contents of acetylcholine (ACh), SOD, MDA and NO in hippocampus and the activity of choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) was detected by ELISA. The expression of N-methyl-D-aspartate receptor (NMDAR), glutamine receptor 2 (GluR), calcium/calmodulin dependent protease Ⅱ (CaMK Ⅱ), Caspase-3, Bcl-2 and Bad proteins in hippocampus of model mice were detected by Western blot.Results:The number and morphology of hippocampal neurons were normal, nucleus, mitochondria, rough endoplasmic reticulum and synaptic membrane of hippocampal neurons in high-dose Naringin group were clear. Compared with the model group, the latency of mice in the high-dose Naringin group was prolonged and the number of errors was reduced ( P<0.01). The levels of MDA and NO in hippocampus of mice in the high-dose Naringin group significantly decreased ( P<0.01), and the activity of SOD significantly increased ( P<0.01). The content of ACh (23.682 ± 2.835 μg/mg prot vs. 14.939 ± 2.901 μg/mg prot), ChAT (163.302 ± 21.278 U/g vs. 89.612 ± 11.497 U/g) increased, AChE (0.367 ± 0.015 U/mg prot vs. 0.471 ± 0.014 U/mg prot) activity decreased ( P<0.01); The expression of Bad (0.441 ± 0.010 vs. 0.633 ± 0.010), Caspase-3 (0.425 ± 0.036 vs. 0.537 ± 0.024) significantly decreased, and the expression of Bcl-2 (0.890 ± 0.014 vs. 0.727 ± 0.009) significantly increased ( P<0.01); The expression of CAMKⅡ (1.043 ± 0.037 vs. 1.475 ± 0.043) significantly decreased ( P<0.01), and the expression of NMDAR1 (0.407 ± 0.037 vs. 0.345 ± 0.012), GluR2 (1.125 ± 0.033 vs. 0.664 ± 0.023) significantly increased ( P<0.01). Conclusion:Naringin could play the role of protecing the neuron and improving the cognition of mice with memory consolidation disorder by regulating the balance of ACh and glutamate system and reducing neuronal apoptosis and antioxidant stress.

The mechanism of Shuyu capsules in treating premenstrual dysphoric disorder caused by liver qi depression based on microdialysis technology from the aspect of Glu-GABA metabolic pathway / 国际中医中药杂志

Objective:To explore the mechanism of premenstrual dysphoric disorder (PMDD) caused by liver-qi depression from the aspect of Glu-GABA metabolic pathways.Methods:Thirty-six rats with similar open field scores and regular estrus cycles were divided into blank group, model group, fluoxetine group, Shuyu capsule group, saikosaponin group and inhibitor group according to the random number table method, with 6 rats in each group. Stereotactic hippocampus surgery was performed during the first estrous cycle reception period after the estrus cycle was determined. In the non-receiving period of the third and fourth estrus cycles, the restraint model was constructed, and from the first day of the modeling, rats of the fluoxetine group were given fluoxetine capsules 2.67 mg/kg, while rats of the Shuyu capsule group and saikosaponin group were given Shuyu capsules 0.408 g/kg and saikosaponin 0.72 mg/kg once a day for 5 consecutive days. Rats in the inhibitor group were injected with 20 μl L-malic acid with 5 mmol/L concentration, which is an inhibitor of glutamate decarboxylase (GAD), in the hippocampus on the last day of modeling. After the administration, weighed the rats and carried out open field experiments. During the second and fivth estrus cycles of rats, the extracellular fluid of the hippocampus was collected by microdialysis technology, and the content of Glu and GABA in the dialysate was detected by HPLC-FLD. Results:After 5 days of administration, compared with the model group, the body weight of rats in the Shuyu capsule group, the inhibitor group and the fluoxetine group increased ( P<0.05), and the total score of the open field experiment decreased ( P<0.05); compared with the model group, during the receiving period of the five estrus cycle, the Glu level of the Shuyu capsule group and the inhibitor group decreased ( P<0.05); In the non-receiving period of the fifth estrus cycle, the Shuyu capsule group, Glu level of the fluoxetine group and the saikosaponin group increased, GABA level of Shuyu capsule group, inhibitor group and fluoxetine group decreased ( P<0.05), Glu/GABA level of Shuyu capsule group, fluoxetine group and inhibitor group (1.49 ± 0.13, 1.32 ± 0.33, 3.92 ± 0.79 vs. 0.35 ± 0.48) was higher than that of the model group ( P<0.05). Conclusion:The therapeutic mechanism of Shuyu capsule in the treatment of PMDD caused by liver Qi depression rats may be ascribed to inhibiting GAD from Glu-GABA metabolic pathway.

Therapeutic effect of oral vaccine based on glutamate decarboxylase 65 on streptozotocin-induced type 1 diabetic mice / 中国药科大学学报

@#To investigate the therapeutic effect of oral vaccine based on glutamate decarboxylase 65 (GAD65) on streptozotocin (STZ) -induced type 1 diabetic (T1D) mice, the mice model of T1D was established by intraperitoneal injection of low dose multiple STZ. CTB-GADIII encapsulated with calcium alginate (Ca-Alg-GADIII) was formulated using crosslinking technology with sodium alginate and calcium chloride, and was administered intragastric to T1D mice once a week for 5 consecutive weeks.Blood glucose and body weight of the mice were recorded weekly, and pharmacodynamics against T1D of Ca-Alg-GADIII were investigated by glucose tolerance assay (OGTT) and pancreatic histopathological analysis. The levels of glutamic acid decarboxylase antibody (GADA), and insulin autoantibody (IAA) and related cytokines (IL-4, IFN-γ, TGF-β1) in serum were detected by ELISA, and the CD4 + T cell subsets were detected by flow cytometry. The immunological mechanism of oral vaccine against T1D was preliminarily discussed. The results showed that the disease-related indicators improved in immunized mice: fasting blood glucose improved, glucose tolerance and insulin secretion increased, pancreatic injury decreased, autoantibodies like GADA and IAA titers significantly decreased, and CD4 + T cell immune balance in mesenteric lymph node (MLN) and pancreatic lymph node (PLN) improved to some extent. The results suggest that oral vaccine Ca-Alg-GADIII has some therapeutic effect on STZ-induced T1D mice.

Effect of electro-acupuncture therapy on limb spasm and excitability of motor neurons in stroke rats / 浙江大学学报·医学版

To investigate the effect of electro-acupuncture therapy on limb spasm and excitability of motor neurons in stroke rats. Ischemic stroke model was induced with middle cerebral artery embolization in SD rats. Thirty-three modeled rats were randomly divided into model group, electro-acupuncture group, and baclofen group with 11 rats in each group, and another 10 rats were taken as sham operation group. The electro-acupuncture group and the baclofen group were treated with electro-acupuncture and baclofen tablets respectively. The model group and the sham operation group had no intervention. The neural function was evaluated with Bederson's scale and balance beam test; the muscle tension was measured with electrophysiography; the pathological changes of brain tissue was examined with HE staining; the content of glutamic acid (Glu) and γ-aminobutyric acid (GABA) in rat cerebral cortex was analyze with enzyme linked immunosorbent assay (ELISA) method, the expression of metabotropic glutamate receptor 1a () and γ-aminobutyric acid type B receptor subunit 1 () mRNA were detected with RT-qPCR. Compared with the model group, the neurological function scores of the electro-acupuncture group and the baclofen group showed a downward trend at d7 after operation (all >0.05), and the neurological function scores of the electro-acupuncture group and the baclofen group were significantly decreased at d12 after the operation (all 0.05). Compared with the model group, the electrophysiological results of the electro-acupuncture group and baclofen group were significantly increased after operation (all <0.05). The results of HE staining showed that there was no cell edema and degeneration in the sham operation group, no pyknosis of the nucleus, and no bleeding in the interstitium. Cell edema and degeneration and mesenchymal congestion appeared in the model group. Compared with the model group, the cytoplasmic edema and degeneration and the interstitial bleeding in the electroacupuncture group and the baclofen group were reduced. Compared with sham operation group, the Glu content and the relative expression of mRNA was increased in the model group, electro-acupuncture group and baclofen group, while the GABA content and the relative expression of mRNA decreased (all <0.05). Compared with model group, the Glu content and the relative expression of mRNA in the electro-acupuncture group and baclofen group decreased, and the GABA content and relative expression of mRNA increased (all <0.05). Electro-acupuncture may improve limb spasm after stroke through regulating the expression of Glu and GABA in the cerebral cortex and the excitability of motor neurons in rats.

Synthesis，Characterization and Anti-tumor Effect Evaluation of Novel Ampelopsin Derivative Poly （γ-glutamic Acid）-ampelopsin Conjugate / 中国药房

OBJECTIVE：To desig n and sy nthesize poly （γ-glutamic acid ）-ampelopsin（γ-PGA-AMP），and to characterize it and evaluate its anti-tumor activity in vitro . METHODS ：Synthetic product was produced through an esterification reaction between γ-PGA and ampelopsin. The structure of synthetic product was characterized by the UV spectrophotometry ，Fourier transform infrared（FT-IR）spectroscopy，1H-NMR spectra and the quantitative elemental analysis. The content of ampelopsin in synthetic product was determined by UV absorption spectrometry at 292 nm. Using 5-FU as positive control ，MTT assay was used to determine inhibitory effects of γ-PGA-AMP and ampelopsin on human breast cancer cell MCF- 7，human liver cancer cell HepG 2 and human lung cancer cell A 549. The IC 50 was calculated. RESULTS ：The results showed that the free 7-hydroxyl group of ampelopsin and the a-carboxyl group of γ-polyglutamic acid had been esterified to obtain γ-PGA-AMP；the yield of γ-PGA-AMP was 55.7％，and the content of ampelopsin was 32.3％. The inhibitory effect of γ-PGA-AMP and ampelopsin on MCF- 7，HepG2 and A 549 cells was obvious. IC 50 of γ-PGA-AMP（to 3 above tumor cells ）were 40.19，28.29 and 55.23 μg/mL，those of ampelopsin were 105.30，81.23，130.10 μg/mL，those of 5-FU were 24.72，87.98，30.99 μg/mL，respectively. CONCLUSIONS ：γ-PGA-AMP with anti-tumor effect in vitro is synthesized successfully ，and its anti-tumor effect is stronger than that of ampelopsin.

Effects of leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation / 解放军医学杂志

[Abstract] Objective To observe the effects of the adipocyte hormone leptin on GABA content and receptor expression in hypothalamus of mice with sleep deprivation, and explore the possible mechanisms. Methods Male C57BL/6 mice were randomly divided into three groups (8 each): control group, sleep deprivation (SD) group and leptin supplement (L-SD) group. Mice in control group were set up in a water environment without sleep deprivation, mice in SD group were set up in a "modified multi-platform water environment" to establish a sleep deprivation model, and mice in L-SD group were given leptin 1.3 mg/kg intraperitoneally twice daily in conjunction with sleep deprivation. Seven days after sleep deprivation, the general conditions of mice were observed, body weight was measured and hypothalamic tissues and plasma specimens were collected. ELISA was used to detect the plasma leptin levels, hypothalamic γ-aminobutyric acid (GABA) and glutamate (Glu) contents. Western blotting was performed to detect the expression levels of GABA key glutamate decarboxylase 67 (GAD67) and GABAA receptor α1 subtype protein (GABAARα1). Results Compared with control group, the weight of mice in SD group significantly reduced [(22.03±0.42) g vs. (17.75±0.75) g, P0.05). The hypothalamic Glu levels were obviously higher in SD group [(686.56±10.01) ng/g] and L-SD group [(668.64+9.93) ng/g] than that in control group [(577.11±16.36) ng/g] (P0.05). The expressive levels of GAD67 and GABAARα1 protein in the hypothalamus of mice in SD group [0.68±0.06, 0.69±0.07] were significantly lower than that in control group (1.09±0.13, 0.99±0.07) (P<0.05); While the expressive levels of GAD67 and GABAARα1 proteins in the hypothalamus of mice in L-SD group (1.39±0.19 and 1.33±0.14, respectively) were significantly higher than those in SD group and control group (P<0.05). Conclusion Leptin can up-regulate the expression of the key GABA synthase GAD67, increase the content of GABA and the expression of GABAARα1 protein in hypothalamus of sleep-deprived mice, which may be an important mechanism of leptin affecting sleep.

Effect of key notes of TCA cycle on L-glutamate production / 生物工程学报

Glutamic acid is an important amino acid with wide range of applications and huge market demand. Therefore, by performing transcriptome sequencing and re-sequencing analysis on Corynebacterium glutamicum E01 and high glutamate-producing strain C. glutamicum G01, we identified and selected genes with significant differences in transcription and gene levels in the central metabolic pathway that may have greatly influenced glutamate synthesis and further increased glutamic acid yield. The oxaloacetate node and α-ketoglutarate node play an important role in glutamate synthesis. The oxaloacetate node and α-ketoglutarate node were studied to explore effect on glutamate production. Based on the integrated strain constructed from the above experimental results, the growth rate in a 5-L fermenter was slightly lower than that of the original strain, but the glutamic acid yield after 48 h reached (136.1±5.53) g/L, higher than the original strain (93.53±4.52) g/L, an increase by 45.5%; sugar-acid conversion rate reached 58.9%, an increase of 13.7% compared to 45.2% of the original strain. The application of the above experimental strategy improved the glutamic acid yield and the sugar-acid conversion rate, and provided a theoretical basis for the metabolic engineering of Corynebacterium glutamicum.

Effect of Modified Suanzaoren Tang on Expression of Glutamate Receptor in Hippocampus of Chronic Restraibt Stress Depression Model Rat / 中国实验方剂学杂志

Objective:To study the effect of modified Suanzaoren Tang on the expression of excitatory amino acids receptor（EAARs） in hippocampus of rats with chronic depression， and to explore the anti-depressant mechanism of modified Suanzaoren Tang based on excitatory amino acids receptor. Method:Sixty SD rats were randomly divided into normal group，model group，and low，middle and high-dose modified Suanzaoren Tang groups，and ketamine group，with 10 rats in each group．Except normal group，the depression model of rats was prepared by using chronic restraint stress（CRS）.The normal group and model group were intragastrically（ig） given normal saline．the modified Suanzaoren Tang groups were intragastrically given corresponding herbal drugs 6，12，24 g·kg-1， ketamine group group were given ketamine 0.015 g·kg-1 through intraoeritoneal injection，for 21 days，once a day．Then the depressive behaviors of rats were observed by Morris water maze and novelty feeding experiment.Western blot was used to detect the levels of DAR1，NMDAR2A，NMDAR2B，GluR1，mGluR1，CaMKⅡα and CaMKⅡβ protein expression in rat hippocampus tissue. Result:Compared with normal group，the time of novel ingestion and escape latencywere prolonged significantly（P<0.01）， and the time of space exploration was shortened significantly（P<0.01）.The levels of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ expression were increased significantly（P<0.01），while the levels of GluR1 and CaMKⅡα expression were decreased significantly（P<0.01）in model group. Compared with model group，the time of novel ingestion and escape latency were shortened significantly （P<0.01）， and the time of space exploration was prolonged significantly（P<0.01）.The levels of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ protein expression were decreased significantly（P<0.01），but the levels of GluR1 and CaMKⅡα expression were increased decreased significantly（P<0.01）in middle and high-dose modified Suanzaoren Tang groups. Conclusion:Modified Suanzaoren Tang can improve the behavior of chronic depression rats effectly. Its mechanism may be related with reduction the expression of NMDAR1，NMDAR2A，NMDAR2B，mGluR1 and CaMKⅡβ protein ，increase the expression of GluR1and CaMKⅡα protein.

Screening of different Pseudomonas and Bacillus Spp. for production of L-glutamic acid and impact of their cell-free filtrate on growth and yield of brinjal (Solanum melongena)

Amino acids can play a different role in plants such as nitrogen source, hormonal precursor, and stress reducingagents. L-glutamic acid is involved in several plant metabolic processes. The objectives of present work were toevaluate the L-glutamic acid production by different agriculturally important Pseudomonas and Bacillus speciesand to determine the effects of L-glutamic acid containing cell-free filtrate on the growth and yield of brinjal. Anexperiment was conducted in a completely randomized block design. Out of eight different strains of Pseudomonas,the highest L glutamic acid was detected in Pseudomonas fluorescens 128 (1.397 g/l) followed by P. fluorescensNSPL07 (1.073 g/l) and Pseudomonas striata RCOF153 (0.563 g/l). Similarly, out of six different species ofBacillus, moderate L-glutamic acid was detected in Bacillus amyloliquefaciens MTCC10439. (0.232 g/l). Thegrowth stimulatory effects of L glutamic acid containing filtrate (200 ppm) were also studied and it was found that allgrowth parameters of brinjal (plant height, fruits per plant, fruit yield, etc.) improved significantly. Results indicatedthat bacterial secretion containing L-glutamic acid along with other amino acids has biostimulatory effects and itshould be used to enhance the plant growth and yield.

Preliminary analysis of primary and secondary factors of γ-aminobutyric acid enrichment in fermentation process of Sojae Semen Praeparatum / 中草药

Objective: To study the primary and secondary factors of γ-aminobutyric acid (GABA) enrichment in the processing of Sojae Semen Praeparatum (SSP), and lay the foundation for revealing the formation mechanism of high content of GABA in SSP. Methods: The dynamic changes of pH value, temperature, moisture, protease and glutamic acid decarboxylase (GAD) in the processing of SSP were determined by conventional methods. The GABA content of each sample was determined by pre-column on-line derivatization established by our laboratory. The correlation between each indicator and GABA was analyzed by SPSS software. Results: Correlation analysis and multiple regression analysis showed that the correlation coefficient between water, acid protease and GABA was 0.211 and -0.340, respectively, and the P values were 0.324 and 0.228, respectively. The correlation was small and there was no statistical difference. The absolute value of the regression coefficient showed that the primary and secondary status of other indicators in the formation of GABA was pH value (-0.375) > temperature (-0.284) > GAD (0.140) > alkaline protease (0.047) > neu-protease (-0.030), in which pH value, temperature and neutral protease had a negative correlation with GABA, and GAD activity and alkaline protease had a positive correlation with GABA. Conclusion: The temperature, pH value, GAD, neutral and alkaline protease are important factors affecting the formation of GABA in the fermentation process of SSP.

Dose-effect relationship between L-glutamic acid and duodenal movement in the vagus pathway / 解剖学报

Objective: To establish a mathematical model of the injection by dorsal motor nucleus of vagus (DMV), the dose of L-glutamic acid (L-Glu) and the duodenal myoelectric activity index of female rabbits was established to provide a theoretical basis for studying the mechanism of action of hormones, cytokines and other vagal activity. Methods: In this experiment, we used microinjection technique to inject L-Glu to DMV by doses of 0 mol/L, 0.05 mol/L, 0.10 mol/L, 0.15 mol/L and 0.20 mol/L, monitoring the duodenal myoelectric activity of ovarian abducted rabbits, the number of samples per dose group was 5, and construct a mathematical model between the dose of L-Glu and the index of duodenal myoelectric activity. Results: After t-test analysis, the amplitude of myoelectric activity increased gradually between adjacent dose groups, and the difference was extremely significant (P < 0.01). The frequency was gradually strengthened, and the difference between 0 mol/L and 0.05 mol/L, 0.10 mol/L and 0.15 mol/L was significant (P < 0.05). The index of myoelectric activity increased gradually, and the difference between groups was extremely significant (P<0.01). With L-Glu concentration as the independent variable x, the duodenal myoelectric activity index was the dependent variable y, the constructor relationship was y= 13.71/1 + exp [-22.35 x (x-0.082)], function fitting accuracy was R =0.9948, P<0.01.belonging to growth type S logic function. Function analysis showed that the L-Glu dose of 0.082 mol/L was the inflection point of the logic function. As the dose range of L-Glu was 0 mol/L-0.082 mol/L, duodenal myoelectric activity index showed an exponential growth pattern, as the L-Glu dose was greater than 0.082 mol/L, the duodenal myoelectric activity index showed a logarithmic growth pattern with a theoretical limit of 13.71. Conclusion: L-Glu has a significant dose-effect relationship on the promotion of duodenal myoelectric activity in rabbits by DMV, and have the effect of interval, the mathematical model laid the theoretical foundation for further research on the role of hormones and cytokines on this basis.

Anxiolytic Effect of Chanmu Anshen Granule and Its Bioactive Mechanism / 中国实验方剂学杂志

Objective:To assess the anxiolytic effect of Chaimu Anshen granules (CMASG) and investigate its bioactive mechanism. Method:ICR mice were randomly divided into normal group, diazepam group(0.002 g·kg-1),Jieyu Anshen granules group(0.001 4 g·kg-1), high, medium, and low-dose (0.001 98,0.000 99,0.000 495 g·kg-1)Chaimu Anshen granule groups, with 20 mice in each group. To detect the anxiolytic effect of CMASG, mice were intragastrically administered for 4 weeks in the morning, and light-dark box transition test and open field test were performed once the other day. After the behavior tests, blood samples were collected. Six mice of each group were perfused with formalin through heart, and then the brains were fixed for immunohistochemistry test. Hippocampus of the other mice in each group were collected and stored in liquid nitrogen. The content of γ-aminobutyric acid(GABA)and glutamic acid(Glu)in hippocampus and blood samples were detected by enzyme-linked immunosorbent assay (ELISA), and the ratio of GABA/Glu was calculated. The expression of GABAα1 receptor was evaluated by the immunohistochemistry method. To test the hypnosis effect of CMASG, mice were administered intragastrically for 7 days. The sub-threshold dose of pentobarbital sodium in the sleep experiment was tested. Result:Compared with normal group, the light-dark box transitions test demonstrated that low-dose and medium-dose CMASG groups significantly prolonged the duration in light box(PPPPPPPPPPα1 receptor protein in hippocampus showed that the medium-dose CMASG significantly increased the expression of GABAα1 protein. The sub-threshold dose of pentobarbital sodium on sleep experiments confirmed that the medium-dose CMASG significantly increased the rate of sleep in mice. Conclusion:CMASG showed an anxiolytic effect, and its bioactive mechanism was related with the increase of GABA content, and the decrease of Glu content in hippocampus. Furthermore, it increased the expression of GABAα1 protein in hippocampus. The changes in content of GABA and Glu in peripheral blood were positively correlated with the changes in hippocampal tissues, which provided reference for clinical diagnosis. CMASG also exhibited an effect in improvement of sleep.

Synthesis and evaluation of a dendritic poly(L-glutamic acid)-graft-polyethylenimine copolymer as an efficient gene delivery vector / 药学学报

The study was designed to synthesize a novel dendritic copolymer composed of polyamidoamine dendrimer G0 as the inner core and poly(L-glutamic acid) grafted low molecular weight polyethylenimine (PGLP) as surrounding arms for gene delivery vector. The molecular structure of PGLP was confirmed by 1H NMR (proton nuclear magnetic resonance spectroscopy). The DNA combination capability of PGLP was examined by gel retardation electrophoresis. The particle sizes and zeta potentials of PGLP/pDNA complexes were determined by dynamic light scattering (DLS). The cytotoxicity of PGLP was evaluated by Cell Counting Kit-8 (CCK-8) and hemolysis assays, which was approved by Research Ethics Committee of the First Affiliated Hospital of Nanchang University. The in vitro transfection efficiency of PGLP was measured by a flow cytometry. The results of physicochemical properties suggested that PGLP could self-assemble with DNA to form complexes with average particle sizes of about 105-200 nm and zeta potentials of about +10 - +28 mV, which could protect DNA from serum degradation. The results of biological properties suggested that PGLP showed more higher transfection efficiencies but lower cytotoxicity than PEI 25K or Lipofectamine 2000 in various cell lines (HEK 293T, HeLa, BEL 7402, RASMC). Importantly, it was found that PGLP/pDNA complexes at w/w = 8 showed more strong serum-resistant capacity than PEI 25K/pDNA complexes. Therefore, PGLP is a promising candidate vector for gene delivery.

Safety and photochemotherapeutic application of poly(-glutamic acid)-based biopolymeric nanoparticle

The safety of nanomaterials, a crucial consideration for clinical translation, is enhanced by using building blocks that are biologically nontoxic. Here, we used poly(-glutamic acid) (-PGA) and dopamine as building blocks of polymeric nanomaterials for carrying hydrophobic anticancer drugs. The introduction of phenylalanine onto -PGA enabled the resulting amphiphilic derivative of -PGA acid to self-assemble in the presence of the anticancer drug paclitaxel (PTX) to form PTX-encapsulated micelles. The surfaces of PTX-loaded micelles were then coated with polymerized dopamine (PDA). The PDA-coated, amphiphilic -PGA-based micelles (AM) carrying PTX (PDA/AM/P) exerted near-infrared-responsive photothermal effects. Near-infrared irradiation of cancer cells treated with PDA/AM/P nanoparticles produced a greater anticancer effect than that observed in other treatment groups, indicating a synergistic effect. Intravenous administration of PDA/AM/P completely ablated tumors and prevented their recurrence. Notably, the safety profile of PDA/AM/P nanoparticles allowed PTX to be delivered at a 3.6-fold higher dose than was possible with PTX solubilized in surfactant, and circumvented the side effects of the surfactant. These results support the multifunctional potential of PDA/AM for the delivery of various hydrophobic drugs and imaging dyes for safe translation of nanomaterials into the clinic.

Regulation of Allergic Immune Responses by Microbial Metabolites

Emerging evidence demonstrates that the microbiota plays an essential role in shaping the development and function of host immune responses. A variety of environmental stimuli, including foods and commensals, are recognized by the host through the epithelium, acting as a physical barrier. Two allergic diseases, atopic dermatitis and food allergy, are closely linked to the microbiota, because inflammatory responses occur on the epidermal border. The microbiota generates metabolites such as short-chain fatty acids and poly-γ-glutamic acid (γPGA), which can modulate host immune responses. Here, we review how microbial metabolites can regulate allergic immune responses. Furthermore, we focus on the effect of γPGA on allergic T helper (Th) 2 responses and its therapeutic application.