[Moxibustion improves learning-memory ability by promoting cellular autophagy and regulating autophagy-related proteins in hippocampus and cerebral cortex in APP/PS1 transgenic Alzheimer's disease mice].

OBJECTIVE: To observe the effect of moxibustion of acupoints of the Governor Vessel on the levels of cellular autophagy, ß amyloid protein (Aß) immunoactivity, and expression of LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins in the hippocampal tissue of APPswe/PS1de9 (APP/PS1) double-transgenic Alzheimer's disease (AD) mice, so as to reveal its underlying mechanisms in improving AD. METHODS: APP/PS1 double-transgenic AD mice were randomly divided into AD model, moxibustion, autophagy-inducer (Rapamycin) and autophagy-inhibitor (3-MA)＋moxibustion groups (n＝10 in each group), and other 10 C57BL/6J male mice (the same age) were used as the normal control group. Herbal-cake (made of Chuanwu [Radix Aconiti Praeparata]) partitioned moxibustion was applied to "Baihui"(GV20), moxibustion was applied to "Fengfu"(GV16) and "Dazhui"(GV14), all for 20 min, once daily for 2 weeks, with one day's off between two weeks. For mice of the autophagy-inducer and 3-MA＋moxibustion groups, Rapamycin (2 mg•kg－1•d－1) and 3-MA (1.5 mg•kg－1•d－1) were separately administered by intraperitoneal injection for 2 weeks. The cognitive ability was examined by Morris water maze tests, and the ultrastructural changes (including autophagic lysosomes, etc.) of hippocampal neurons were observed by using transmission electron microscopy. The immunoactivity of cerebral cortex and hippocampal Amyloid ß peptide 1-42 (Aß1-42) was detected by immunohistochemistry, and the expression levels of hippocampal LC3-Ⅰ， LC3-Ⅱ， p62 and p-P70S6K proteins were detected by Western blot. RESULTS: After modeling, the escape latency of Morris water maze tasks was prolonged in the model group than in the normal control group (P<0.05) and obviously shortened in the moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.05). Results of transmission electron microscope showed deformed, irregular or atrophic neurons with rough and incomplete and fuzzy nuclear membrane, and decreased intracellular autophagosomes in the hippocampus in the model group, and partial irregular, atrophic neurons with more autophagic vesicles and lysosomes in the moxibustion group. The expression levels of Aß1-42 in both cerebral cortex and hippocampus tissues, and LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus were consi-derably up-regulated in the model group relevant to the normal control group (P<0.01), and evidently down-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01), while that of hippocampal LC3-Ⅱ protein and LC3-Ⅱ/Ⅰ ratio levels were obviously down-regulated in the model group relevant to the normal control group (P<0.01), and significantly up-regulated in both moxibustion and autophagy-inducer groups (not the autophagy-inhibitor group) than in the model group (P<0.01)．. CONCLUSION: Moxibustion can improve the cognitive ability of APP/PS1 double-transgenic AD mice, which is associated with its effects in promoting hip-pocampal and cerebral cortex autophagy level, and down-regulating the expression levels of Aß1-42, LC3-Ⅰ， p62 and p-P70S6K proteins in the hippocampus.

A Single Mutation Increases the Thermostability and Activity of Aspergillus terreus Amine Transaminase.

Enhancing the thermostability of (R)-selective amine transaminases (AT-ATA) will expand its application in the asymmetric synthesis of chiral amines. In this study, mutual information and coevolution networks of ATAs were analyzed by the Mutual Information Server to Infer Coevolution (MISTIC). Subsequently, the amino acids most likely to influence the stability and function of the protein were investigated by alanine scanning and saturation mutagenesis. Four stabilized mutants (L118T, L118A, L118I, and L118V) were successfully obtained. The best mutant, L118T, exhibited an improved thermal stability with a 3.7-fold enhancement in its half-life (t1/2) at 40 °C and a 5.3 °C increase in T5010 compared to the values for the wild-type protein. By the differential scanning fluorimetry (DSF) analysis, the best mutant, L118T, showed a melting temperature (Tm) of 46.4 °C, which corresponded to a 5.0 °C increase relative to the wild-type AT-ATA (41.4 °C). Furthermore, the most stable mutant L118T displayed the highest catalytic efficiency among the four stabilized mutants.

[Moxibustion Improves Learning Ability by Regulating Hippocampal Neurotrophic Factor Expression and Notch Signaling in Vascular Dementia Rats].

OBJECTIVE: To observe the effect of moxibustion on the learning ability and expression of neurotrophic factors and Notch signaling in vascular dementia (VD) rats, so as to explore its neurogenesis mechanism underlying improvement of VD. METHODS: Sixty SD rats were equally and randomly divided into sham operation (sham), model, medication and moxibustion groups (n=15 rats/group). The VD model was established by occlusion of the bilateral cervical common arteries and reperfusion. Moxibustion was applied to "Dazhui"(GV 14), "Guanyuan"(CV 4) and "Mingmen"(GV 4)for 15 minutes, once daily, 6 times a week for 4 weeks. Rats of the medication group were treated by gavage of nimodipine (2 mg·kg-1·d-1),3 times a day, 6 days a week for 4 weeks. Morris water maze tests were performed to detect the rat's learning-memory ability. The infarcted size of the brain was detected by using 2,3,5-triphenyltetrazolium chloride (TTC) staining, and H.E. staining was used to detect the histopathological changes. The expression level of glia fibrillary acidic protein (GFAP), nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), Notch 1 (a receptor), Hes 3 (a downstream effector) mRNAs and proteins in the hippocampal tissues were detected by quantitative real-time PCR and Western blot, respectively. RESULTS: After 4 weeks' intervention, modeling-induced increase of escape latency was significantly shortened in both moxibustion and medication groups relevant to the model group (P<0.05), and the infarct size was reduced and the damage degree of nerve cells in the brain tissue alleviated. The expression levels of BDNF, NGF, GFAP, Hes 3, Notch 1 genes and proteins were significantly up-regulated in the model group relevant to the sham operation group (P<0.05,P<0.01). After the intervention, the expression levels of hippocampal BDNF, NGF, GFAP, Hes 3 and Notch 1 mRNAs and proteins in the moxibustion group, and NGF and GFAP mRNAs, and BDNF, NGF, GFAP, Hes 3 and Notch 1 proteins in the medication group were further obviously up-regulated relevant to the model group (P<0.05, P<0.01). The effect of moxibustion was significantly superior to that of medication in up-regulating GFAP, Hes 3, Notch 1 mRNAs expression (P<0.05,P<0.01). No significant differences were found in the expression of BDNF, Hes 3 and Notch 1 mRNAs in the medication group relevant to the model group (P<0.05), and between the moxibustion and medication groups in up-regulating the expression of BDNF and NGF mRNAs, and in up-regulating the expression of BDNF, NGF, GFAP, Hes 3 and Notch 1 proteins (P<0.05). CONCLUSIONS: Moxibustion can improve learning ability in VD rats, which may be associated with its effects in up-regulating the expression of neurotrophic factors and in potentiating Notch signaling.

Dramatic increases in blood glutamate concentrations are closely related to traumatic brain injury-induced acute lung injury.

Traumatic brain injury-induced acute lung injury (TBI-ALI) is a serious complication after brain injury for which predictive factors are lacking. In this study, we found significantly elevated blood glutamate concentrations in patients with TBI or multiple peripheral trauma (MPT), and patients with more severe injuries showed higher blood glutamate concentrations and longer durations of elevated levels. Although the increase in amplitude was similar between the two groups, the duration was longer in the patients with TBI. There were no significant differences in blood glutamate concentrations in the patients with MPT with regard to ALI status, but the blood glutamate levels were significantly higher in the patients with TBI-ALI than in those without ALI. Moreover, compared to patients without ALI, patients with TBI showed a clearly enhanced inflammatory response that was closely correlated with the blood glutamate levels. The blood glutamate concentration was also found to be a risk factor (adjusted odds ratio, 2.229; 95% CI, 1.082-2.634) and was a better predictor of TBI-ALI than the Glasgow Coma Scale (GCS) score. These results indicated that dramatically increased blood glutamate concentrations were closely related to the occurrence of TBI-ALI and could be used as a predictive marker for "at-risk" patients.