Histone deacetylase inhibitors inhibit cervical cancer growth through Parkin acetylation-mediated mitophagy

Parkin, an E3 ubiquitin ligase, plays a role in maintaining mitochondrial homeostasis through targeting damaged mitochondria for mitophagy. Accumulating evidence suggests that the acetylation modification of the key mitophagy machinery influences mitophagy level, but the underlying mechanism is poorly understood. Here, our study demonstrated that inhibition of histone deacetylase (HDAC) by treatment of HDACis activates mitophagy through mediating Parkin acetylation, leading to inhibition of cervical cancer cell proliferation. Bioinformatics analysis shows that Parkin expression is inversely correlated with HDAC2 expression in human cervical cancer, indicating the low acetylation level of Parkin. Using mass spectrometry, Parkin is identified to interact with two upstream molecules, acetylase acetyl-CoA acetyltransferase 1 (ACAT1) and deacetylase HDAC2. Under treatment of suberoylanilide hydroxamic acid (SAHA), Parkin is acetylated at lysine residues 129, 220 and 349, located in different domains of Parkin protein. In in vitro experiments, combined mutation of Parkin largely attenuate the interaction of Parkin with PTEN induced putative kinase 1 (PINK1) and the function of Parkin in mitophagy induction and tumor suppression. In tumor xenografts, the expression of mutant Parkin impairs the tumor suppressive effect of Parkin and decreases the anticancer activity of SAHA. Our results reveal an acetylation-dependent regulatory mechanism governing Parkin in mitophagy and cervical carcinogenesis, which offers a new mitophagy modulation strategy for cancer therapy.

Overexpression of NAT10 induced platinum drugs resistance in breast cancer cell / 中华肿瘤杂志

Objective: To observe the platinum drugs resistance effect of N-acetyltransferase 10 (NAT10) overexpression in breast cancer cell line and elucidate the underlining mechanisms. Methods: The experiment was divided into wild-type (MCF-7 wild-type cells without any treatment) group, NAT10 overexpression group (H-NAT10 plasmid transfected into MCF-7 cells) and NAT10 knockdown group (SH-NAT10 plasmid transfected into MCF-7 cells). The invasion was detected by Transwell array, the interaction between NAT10 and PARP1 was detected by co-immunoprecipitation. The impact of NAT10 overexpression or knockdown on the acetylation level of PARP1 and its half-life was also determined. Immunostaining and IP array were used to detect the recruitment of DNA damage repair protein by acetylated PARP1. Flow cytometry was used to detect the cell apoptosis. Results: Transwell invasion assay showed that the number of cell invasion was 483.00±46.90 in the NAT10 overexpression group, 469.00±40.50 in the NAT10 knockdown group, and 445.00±35.50 in the MCF-7 wild-type cells, and the differences were not statistically significant (P>0.05). In the presence of 10 μmol/L oxaliplatin, the number of cell invasion was 502.00±45.60 in the NAT10 overexpression group and 105.00±20.50 in the NAT10 knockdown group, both statistically significant (P<0.05) compared with 219.00±31.50 in wild-type cells. In the presence of 10 μmol/L oxaliplatin, NAT10 overexpression enhanced the binding of PARP1 to NAT10 compared with wild-type cells, whereas the use of the NAT10 inhibitor Remodelin inhibited the mutual binding of the two. Overexpression of NAT10 induced PARP1 acetylation followed by increased PARP1 binding to XRCC1, and knockdown of NAT10 expression reduced PARP1 binding to XRCC1. Overexpression of NAT10 enhanced PARP1 binding to LIG3, while knockdown of NAT10 expression decreased PARP1 binding to LIG3. In 10 μmol/L oxaliplatin-treated cells, the γH2AX expression level was 0.38±0.02 in NAT10 overexpressing cells and 1.36±0.15 in NAT10 knockdown cells, both statistically significant (P<0.05) compared with 1.00±0.00 in wild-type cells. In 10 μmol/L oxaliplatin treated cells, the apoptosis rate was (6.54±0.68)% in the NAT10 overexpression group and (12.98±2.54)% in the NAT10 knockdown group, both of which were statistically significant (P<0.05) compared with (9.67±0.37)% in wild-type cells. Conclusion: NAT10 overexpression enhances the binding of NAT10 to PARP1 and promotes the acetylation of PARP1, which in turn prolongs the half-life of PARP1, thus enhancing PARP1 recruitment of DNA damage repair related proteins to the damage sites, promoting DNA damage repair and ultimately the survival of breast cancer cells.

Acetilación N-terminal, acetiltransferasas y mutaciones en subunidad auxiliar NAA15 de N-terminal acetiltransferasa A, asociadas a discapacidad intelectual y trastorno del espectro autista: una revisión bibliográfica / N-terminal acetylation, acetyltransferases and variants in NAA15 auxiliary subunit of the N-terminal acetyltransferase A associated with intellectual disability and autism spectrum disorder: a literature review

Las N-terminal acetiltransferasas (NaT) son fundamentales en el desarrollo, funcionamiento y vida media celular, acetilando gran parte del proteoma humano. Entre las ocho NaT identificadas, N-terminal acetiltransferasa A (NaTA) acetila a un mayor número de sustratos, teniendo además un rol fundamental en el neurodesarrollo. Previamente, estudios han demostrado que mutaciones en la subunidad catalítica de NaTA, NAA10, se asocian con trastornos del neurodesarrollo. Sin embargo, nuevas líneas investigativas sugieren que mutaciones de la subunidad auxiliar, NAA15, también tendrían un rol importante en el desarrollo de estos trastornos. Esta revisión se realiza con el objetivo de recopilar evidencia sobre variantes de NAA15 relacionadas con Discapacidad Intelectual (DI) y Trastorno de Espectro Autista (TEA). Se consultaron fuentes actualizadas sobre acetilación N-terminal, NaT, DI y TEA y mutaciones reportadas de NAA15 y sus expresiones fenotípicas, publicadas entre 2011 y 2022. Se concluye que, aun cuando existe relación entre mutaciones de NAA15, DI y TEA, todavía es necesario esclarecer los mecanismos fisiopatológicos de estos trastornos, el rol de NaTA y el impacto de variantes de sus subunidades en las vías moleculares y el fenotipo, lo que se dificulta por razones que van desde la complejidad de estas vías hasta el elevado costo de análisis genéticos. Se sugiere continuar la investigación en esta área, para comprender las bases moleculares subyacentes a estos trastornos y el rol de las mutaciones en subunidades de NaTA, con el fin último de estudiar potenciales tratamientos que mejoren la calidad de vida de las personas con estos trastornos y sus familias.

Nt-acetyltransferases (NaT) are essential in cell development, function and half-life, catalyzing most of the human proteome. Among the eight NaTs identified, N-terminal acetyltransferase A (NaTA) acetylates a greater number of substrates, also having a fundamental role in neurodevelopment. Previously, studies have shown that mutations in the catalytic subunit of NaTA, NAA10, are associated with neurodevelopmental disorders. However, new research lines suggest that mutations of the NAA15 helper subunit also plays an important role in the development of these disorders. This review is carried out with the objective of gathering evidence on NAA15 variants related to Intellectual Disability (ID) and Autism Spectrum Disorder (ASD). Updated sources on N-terminal acetylation, N-acetyltransferases, DI and TEA and reported mutations of NAA15 and their phenotypic expressions, published between 2011 and 2022 were consulted. It is concluded that even though there is a relationship between mutations of NAA15, ID and ASD exists, it is still necessary to clarify the pathophysiological mechanisms of these disorders, the role of NaTA and the impact of variants of its subunits in the molecular pathways and in the phenotype, for reasons ranging from the complexity of these pathways to the high cost of genetic testing. It is suggested to continue research in this area, to understand the molecular bases underlying these disorders and the role of mutations in NatA subunits, with the ultimate aim of studying potential treatments that improve the quality of life of people with these disorders and their families.

Role of cytochrome P450 1A2 and N-acetyltransferase 2 in 2, 6-dimethylaniline induced genotoxicity

Abstract The purpose of the current work was to assess a possible role of cytochrome P450 1A2 (CYP1A2) and N-acetyltransferase 2 (NAT2) in the metabolic activation of 2,6-dimethylaniline (2,6-DMA) and also clarify the function of DNA repair in affecting the ultimate mutagenic potency. Two cell lines, nucleotide excision repair (NER)-deficient 5P3NAT2 and proficient 5P3NAT2R9 both expressing CYP1A2 and NAT2, were treated with 2,6-DMA for 48 h or its metabolites for 1 h. Cell survival determined by trypan blue exclusion and MTT assays, and 8-azaadenine-resistant mutants at the adenine phosphoribosyltransferase (aprt) gene locus were evaluated. 5P3NAT2 and 5P3NAT2R9 cells treated with 2,6-DMA and its metabolites showed a dose-dependent increase in cytotoxicity and mutant fraction; N-OH-2,6-DMA and 2,6-DMAP in serum-free α-minimal essential medium (MEM) are more potent than 2,6-DMA in complete MEM. 5P3NAT2 cells was more sensitive to the cytotoxic and mutagenic action than 5P3NAT2R9 cells. H2DCFH-DA assay showed dose-dependent ROS production under 2,6- DMAP treatment. These findings indicate that the genotoxic effects of 2,6-DMA are mediated by CYP1A2 activation via N-hydroxylation and the subsequent esterification by the phase II conjugation enzyme NAT2, and through the generation of ROS by hydroxylamine and/or aminophenol metabolites. NER status is also an important contributor

Homozygotes NAT2*5B slow acetylators are highly associated with hepatotoxicity induced by anti-tuberculosis drugs

BACKGROUND Distinct N-acetyltransferase 2 (NAT2) slow acetylators genotypes have been associated with a higher risk to develop anti-tuberculosis drug-induced hepatotoxicity (DIH). However, studies have not pointed the relevance of different acetylation phenotypes presented by homozygotes and compound heterozygotes slow acetylators on a clinical basis. OBJECTIVES This study aimed to investigate the association between NAT2 genotypes and the risk of developing DIH in Brazilian patients undergoing tuberculosis treatment, focusing on the discrimination of homozygotes and compound heterozygotes slow acetylators. METHODS/FINDINGS The frequency of NAT2 genotypes was analysed by DNA sequencing in 162 patients undergoing tuberculosis therapy. The mutation analyses revealed 15 variants, plus two new NAT2 mutations, that computational simulations predicted to cause structural perturbations in the protein. The multivariate statistical analysis revealed that carriers of NAT2*5/*5 slow acetylator genotype presented a higher risk of developing anti-tuberculosis DIH, on a clinical basis, when compared to the compound heterozygotes presenting NAT2*5 and any other slow acetylator haplotype [aOR 4.97, 95% confidence interval (CI) 1.47-16.82, p = 0.01]. CONCLUSION These findings suggest that patients with TB diagnosis who present the NAT2*5B/*5B genotype should be properly identified and more carefully monitored until treatment outcome in order to prevent the occurrence of anti-tuberculosis DIH.

Relationship between choline acetyltransferase positive neurons in parabrachial nucleus and development of fear memory in mice / 中华麻醉学杂志

Objective:To evaluate the relationship between choline acetyltransferase (ChAT) positive neurons in parabrachial nucleus and development of fear memory in mice.Methods:Eighteen healthy male ChAT-ires-cre mice, aged 8-9 weeks, weighing 22-25 g, were divided into 3 groups ( n=6 each) using a random number table method: Cre-dependent AAV-DIO-hM 3Dq-mcherry (Gq) virus/clozapine-N-oxide (CNO) group (group Gq/CNO), Gq/normal saline (NS) group (group Gq/NS) and Cre-dependent AAV-DIO-mcherry (mc) virus/CNO group (group mc/CNO). Gq virus was injected into parabrachial nucleus, and CNO 2 mg/kg was injected intraperitoneally 3 weeks later in group Gq/CNO.Gq virus was injected into parabrachial nucleus, and the equal volume of normal saline was injected intraperitoneally 3 weeks later in group Gq/NS.In group mc/CNO, mc virus was injected into parabrachial nucleus, and CNO 2 mg/kg was injected intraperitoneally 3 weeks later.The fear conditioning test was performed at 30 min after intraperitoneal injection in all the 3 groups.The brains were then removed and sliced.The virus expression and areas of the brain projected by ChAT positive neurons were observed. Results:Compared with group Gq/CNO, the percentage of freezing time was significantly increased during testing phase in Gq/NS and mc/CNO groups ( P<0.05). Gq/mc virus carrying fluorescent protein mcherry was expressed in parabrachial nucleus and was co-expressed with mcherry-ChAT.The fibers of ChAT positive neurons projected to the red nucleus, substantia nigra, central amygdala, anterodorsal thalamic nucleus and bed nucleus of stria terminalis. Conclusion:The ChAT positive neurons in parabrachial nucleus are involved in the regulation of the development of fear memory in mice, which can impair fear memory, and the regulation is carried out probably through central amygdala.

Expression of histone acetyltransferase P300 in hepatocellular carcinoma tissue and its clinical significance / 国际肿瘤学杂志

Objective:To investigate the expression and clinical significance of histone acetyltransferase P300 in hepatocellular carcinoma.Methods:From January 2013 to December 2017, surgical specimens of 100 patients with hepatocellular carcinoma were collected from the Department of General Surgery of Sichuan Mianyang 404 Hospital. The expressions of P300, CD90, alpha fetoprotein (AFP), Ki-67 and CD34 in hepatocellular carcinoma tissue were detected. At the same time, 42 hepatic hemangioma specimens and 56 liver tissue specimens with moderate to severe liver cirrhosis were collected, and the positive expression rate of P300 in tissues was detected. The correlations between the expression of P300 and clinicopathological features and prognosis of patients with hepatocellular carcinoma were analyzed.Results:The positive expression rates of P300 in normal liver tissue, liver cirrhosis tissue and hepatocellular carcinoma tissue increased gradually, which were 11.9% (5/42), 32.1% (18/56) and 57.0% (57/100) respectively, with a statistically significant difference ( χ2=27.192, P<0.001). Tumor grade ( χ2=9.337, P=0.009), T stage ( χ2=8.794, P=0.032), clinical TNM stage ( χ2=6.121, P=0.013), AFP ( χ2=11.040, P=0.001), CD90 ( χ2=9.903, P=0.002), CD34 ( χ2=4.066, P=0.044) significantly affected the expression of P300. Spearman rank correlation analysis showed that the abnormal expression of P300 was positively correlated with the expression of AFP ( r=0.335, P=0.001), CD90 ( r=0.328, P=0.002) and CD34 ( r=0.264, P=0.047) , but had no significant correlation with the expression of Ki-67 ( P>0.05). Survival analysis showed that the 5-year survival rate of patients with P300 positive expression was 17.6%, and that of patients with P300 negative expression was 62.5%, and there was a statistically significant difference ( χ2=10.596, P<0.001). Cox multivariate analysis showed that P300 positive expression ( RR=2.554, 95% CI: 1.261-4.502, P=0.009), CD90 positive expression ( RR=3.574, 95% CI: 1.021-11.980, P=0.030) and TNM Ⅱ-Ⅳ stage ( RR=0.332, 95% CI: 0.105-0.596, P=0.002) were independent risk factors for poor prognosis of patients with hepatocellular carcinoma. Conclusion:The positive expression of P300 is closely related to the occurrence of hepatocellular carcinoma and can be used as an independent factor to judge the poor prognosis of patients with hepatocellular carcinoma.

Regulation of Hypnotic Active Components of Cnidii Fructus on Melatonin Synthesis Rate-limiting Enzyme AANAT in Rats with PCPA-induced Insomnia / 中国实验方剂学杂志

Objective:To observe the effects of Cnidii Fructus hypnotic active components （CHC） on the behaviors of rats with p-chlorophenylalanine （PCPA）-induced insomnia and melatonin （MT） synthesis rate-limiting enzyme arylalkylamine <italic>N</italic>-acetyltransferase （AANAT）， and explore the protective mechanism of CHC on the pineal gland. Method:Male SD rats of SPF grade were randomly divided into a blank control group， a model group， a MT group， and high-， medium-， and low-dose CHC groups with 10 rats in each group. Except for the blank control group， other groups received 4.5% PCPA suspension at 10 mL·kg^-1， intragastric administration， for two consecutive days. After PCPA model of insomnia was established， normal and model groups were gavaged at the same volume of 2% Tween-80， MT control group （10 mg·kg^-1）， CHC was high， medium and low （60， 30， 15 mg·kg^-1）， 10 mL·kg^-1， once a day， for consecutive 7 days. Four days after administration， open field， elevated cross maze， and pentobarbital sodium-induced sleep tests were conducted， respectively. Serum MT was detected by enzyme-linked immunosorbent assay. The mRNA expression level of AANAT was determined by real-time fluorescence-based quantitative polymerase chain reaction （Real-time PCR）. The expression of AANAT protein in the pineal gland was detected by Western blot. Result:Compared with the results in the blank control group， the total distance of open field activity and standing times and duration in the central area were increased （<italic>P</italic><0.05， <italic>P</italic><0.01）， the proportions of open arm entry （OE%） and open arm time （OT%） were decreased （<italic>P</italic><0.05）， and the sleep latency was prolonged （<italic>P</italic><0.01） in the model group. Compared with the model group， no significant difference was observed in the low-dose CHC group， while other groups exhibited reduced total distance of activity （<italic>P</italic><0.05， <italic>P</italic><0.01）， elevated OE% （<italic>P</italic><0.05）， shortened sleep latency， and prolonged sleep time （<italic>P</italic><0.05， <italic>P</italic><0.01）. Compared with the serum MT in the blank control group， that in the model group was decreased （<italic>P</italic><0.01）. Compared with the model group， no significant difference was observed in the low-dose CHC group， while other groups displayed increased serum MT （<italic>P</italic><0.05）. The mRNA and protein expression of AANAT was decreased in the model group as compared with that in the blank control group （<italic>P</italic><0.01）. Compared with the model group， the MT group and the high-dose CHC group showed up-regulated expression （<italic>P</italic><0.05）. Conclusion:CHC improved the behavioral indexes of PCPA-induced insomnia， increased the synthesis and secretion of MT in pineal cells， and elevated the serum MT level， which was related to the up-regulation of the mRNA and protein expression of AANAT in the pineal gland.

Molecular Mechanism of Salvia miltiorrhiza Quality Formation Based on Single Nucleotide Polymorphism of Functional Genes / 中国实验方剂学杂志

Objective:To investigate the relationship between the single nucleotide polymorphism（SNP）of function genes and effective components of <italic>Salvia miltiorrhiza</italic> and the molecular mechanism of specific quality formation of <italic>S. miltiorrhiza</italic>. Method:The fingerprints of components in <italic>S. miltiorrhiza</italic> from eight different habitats and varieties were obtained by high-performance liquid chromatography （HPLC）. The full-length cDNA of three functional genes<italic> </italic>acetyl-CoA C-acetyltransferase（<italic>SmAACT</italic>），4-diphosphocytidyl-2-C-methyl-<italic>D</italic>-erythritol kinase（<italic>SmCMK</italic>） and isopentenyl diphosphate isomerase（<italic>SmIPPI</italic>） in tanshinone metabolic pathway were amplified by polymerase chain reaction（PCR），cloned， and sequenced，followed by bioinformatics analysis. Result:The full-length cDNA sequences of three functional genes <italic>SmAACT</italic>，<italic>SmCMK</italic>， and <italic>SmIPPI</italic> in tanshinone metabolic pathway were obtained from 23 strains of <italic>S. miltiorrhiza</italic> from eight different habitats and varieties. As revealed by the analysis of SNP and amino acid polymorphisms of three functional genes，18，16， and 14 SNP sites were found respectively. HPLC results showed the samples from Beijing，Hubei，Shandong （No. SDB），Shanxi，Henan， and Shandong （No. SDZ） were clustered into one branch，and those from Hebei and Inner Mongolia were clustered into another branch， which suggested that the variation trend of <italic>S. miltiorrhiza</italic> components had little correlation with geographical distance，but the variety was a critical factor for the quality. Conclusion:There was an obvious genetic differentiation trend in <italic>S. miltiorrhiza</italic> from different habitats，and different origin-specific genotypes were formed. The molecular mechanism of the formation of the specific quality of <italic>S. miltiorrhiza</italic> from different habitats was discussed，which laid a foundation for the stability and effectiveness of clinical medication，and guided the breeding of excellent varieties of <italic>S. miltiorrhiza</italic>.

Construction of acetyl-CoA and DBAT hybrid metabolic pathway for acetylation of 10-deacetylbaccatin III to baccatin III

10-Deacetylbaccatin III (10-DAB) C10 acetylation is an indispensable procedure for Taxol semi-synthesis, which often requires harsh conditions. 10-Deacetylbaccatin III-10-

Protective effect of CD73 inhibitor α, ß-methylene ADP against amyloid-ß-induced cognitive impairment by inhibiting adenosine production in hippocampus

BACKGROUND: Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disease. Recent studies have reported the close association between cognitive function in AD and purinergic receptors in the central nervous system. In the current study, we investigated the effect of CD73 inhibitor α, ß-methylene ADP (APCP) on cognitive impairment of AD in mice, and to explore the potential underlying mechanisms. RESULTS: We found that acute administration of Aß1­42 (i.c.v.) resulted in a significant increase in adenosine release by using microdialysis study. Chronic administration of APCP (10, 30 mg/kg) for 20 d obviously mitigated the spatial working memory impairment of Aß1­42-treated mice in both Morris water maze (MWM) test and Y-maze test. In addition, the extracellular adenosine production in the hippocampus was inhibited by APCP in Aß-treated mice. Further analyses indicated expression of acetyltransferase (ChAT) in hippocampus of mice of was significantly reduced, while acetylcholinesterase (AChE) expression increased, which compared to model group. We observed that APCP did not significantly alter the NLRP3 inflammasome activity in hippocampus, indicating that anti-central inflammation seems not to be involved in APCP effect. CONCLUSIONS: In conclusion, we report for the first time that inhibition of CD73 by APCP was able to protect against memory loss induced by Aß1­42 in mice, which may be due to the decrease of CD73-driven adenosine production in hippocampus. Enhancement of central cholinergic function of the central nervous system may also be involved in the effects of APCP.

Role of PfGCN5 in nutrient sensing and transcriptional regulation in Plasmodium falciparum

Malaria is a deadly, infectious disease caused by the parasite Plasmodium, leading to millions of deathsworldwide. Plasmodium requires a coordinated pattern of sequential gene expression for surviving in bothinvertebrate and vertebrate host environments. As parasites largely depend on host resources, they also developefficient mechanisms to sense and adapt to variable nutrient conditions in the environment and modulate theirvirulence. Earlier we have shown that PfGCN5, a histone acetyltransferase, binds to the stress-responsive andvirulence-related genes in a poised state and regulates their expression under temperature and artemisinintreatment conditions in P. falciparum. In this study, we show upregulation of PfGCN5 upon nutrient stresscondition. With the help of chromatin immunoprecipitation coupled high-throughput sequencing (ChIP-seq)and transcriptomic (RNA-sequencing) analyses, we show that PfGCN5 is associated with the genes that areimportant for the maintenance of parasite cellular homeostasis upon nutrient stress condition. Furthermore, weidentified various metabolic enzymes as interacting partners of PfGCN5 by immunoprecipitation coupled withmass spectroscopy, possibly acting as a sensor of nutrient conditions in the environment. We also demonstratedthat PfGCN5 interacts and acetylates PfGAPDH in vitro. Collectively, our data provides important insights intotranscriptional deregulation upon nutrient stress condition and elucidate the role of PfGCN5 during nutrientstress condition.

Effects of silencing Kat5 gene on proliferation and apoptosis of thyroid papillary carcinoma cells / 吉林大学学报(医学版)

Objective: To investigate the effects of lysine acetyltransferase (Kat5) on the proliferation and apoptosis of thyroid papillary carcinoma cells. Methods: RT-PCR and Western blotting methods were used to determine the expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma BHP10-3. TPC-1 . K1 cells and thyroid epithelial Nthy-ori3-l cells. The thyroid papillary carcinoma TPC-1 cells were divided into blank control group, negative control group and silencing Kat5 group (si-Kat5 group). The cells in blank control group were not transfected. the cells in negative control group were transfected with negative control siRNA. and the cells in si-Kat5 group were transfected with Kat5 siRNA. The expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma TPC-1 cells in various groups were detected by RT-PCR and Western blotting methods. The proliferation activities of the TPC-1 cells in various groups were measured by CCK-8 method, the colony formation assay was used to measure the clone formation rates of the TPC-1 cells in various groups, the apoptotic rates of the TPC-1 cells in various groups were detected by flow cytometry, and the expression levels of cyclin-dependent kinase 2 (CDK2). P21, P53. cleaved caspase-3. P13K, phosphorylated P13K (p-P13K), AKT, and phosphorylated AKT (p-AKT) protein in the TPC-1 cells in various groups were detected by Western blotting method. Results: The expression levels of Kat5 mRNA and protein in the thyroid papillary carcinoma BHP10-3. TPC-1 and K1 cells were higher than those in thyroid epithelial Nthy-ori3-l cells (P<0. 05). The differences in the Kat5 mRNA and protein expression levels, the proliferation activities, the clone formation rates, the apoptotic rates and the expression levels of CDK2. P21. P53. cleaved caspase-3. P13K. p-P13K . AKT and p-AKT proteins in the TPCM cells between various groups were statistically significant (P<0.05). Compared with blank control group and negative control group, the expression levels of Kat5 mRNA and protein in the TPC-1 cells in si-Kat5 group were decreased ( P

Effect of Ultrafine Powder of Gastrodiae Rhizoma in Improving Learning and Memory Ability of Vascular Dementia Rats by Regulating Cholinergic System / 中国实验方剂学杂志

Objective:To investigate whether ultrafine powder of Gastrodiae Rhizoma （UPG） can alleviate the learning and memory impairment of vascular dementia rats and delay the process of VD， and whether this effect is related to the release of acetylcholine （Ach） through the regulation with acetylcholinesterase （AChE） and choline acetyltransferase （ChAT） and control of cholinergic system. Method:SD rats were randomly divided into sham operation group， UPG low dose group （0.45 g·kg-1）， UPG high dose group （1.8 g·kg-1） and Huperzine A group （80 μg·kg-1）， with 12 rats in each group. The drug administration groups were given orally drugs once a day for 8 weeks， and sham group and model group were given orally the same amount of distilled water. The learning and memory ability of the rats with VD were evaluated by the Morris water maze. Htoxylin eosin（HE） staining was used for pathomorphological observation of hippocampus CA1 area of the rats. The content of Ach was determined by enzyme-linked immunosorbent assay（ELISA）， AChE and ChAT protein expressions were detected by Western blot， and expression of ChAT in hippocampus CA1 area was observed by immunohistochemistry. Result:Compared with the sham operation group， the escape latency of the model group was significantly increased （P<0.01）， and the frequency of crossings platform and the time of staying in the target quadrant were reduced significantly （P<0.01）. HE staining of hippocampal tissues from VD rat showed neuron disorders， loss and degeneration and necrosis， pyknosis of the nucleus and light coloration of the cytoplasm. The level of acetylcholine in the hippocampus was significantly decreased by ELISA （P<0.05）， the expression level of AChE protein was significantly up-regulated， and the expression level of ChAT protein was significantly down-regulated （P<0.01）. Compared with model group， each administration group could significantly reduce the escape latency of the model rats， and significantly increase the frequency of crossing platform and the time of staying in the target quadrant （P<0.01）， the content of Ach was significantly increased （P<0.05）， the expression of AChE protein was significantly down-regulated （P<0.01）， while the expression of ChAT protein was significantly up-regulated （P<0.01）. Conclusion:UPG improves the learning and memory ability of vascular dementia rats， and its mechanism may be related to the increase of Ach， ChAT level and the decrease of AChE level.

Analysis of metabolic associated gene DLAT expression in colorectal cancer based on multi-database and its clinical significance / 解放军医学杂志

Objective To screen the metabolic associated genes (MAG) that are related to the survival of colorectal cancer (CRC) patients, investigate the relationship between the expression of dihydrolipoamide S-acetyltransferase (DLAT) and clinical significance and prognosis value in CRC, and establish the DLAT co-expression molecular regulatory network. Methods The cancer genome atlas (TCGA) database combined with whole genome transcript sequencing data and clinical case data were used to do the survival analysis of candidate MAG; The DLAT expression in CRC and the relationship between the expression level and pathological characteristics and prognosis of CRC patients were analyzed through GEO database, Oncomine and KM-plotter platforms. The DLAT co-expression gene lists were searched in cBioPortal and STRING to analyze functional clustering and pathway enrichment, and establish the DLAT co-expression molecular regulatory network. Results Survival analysis in multiple databases indicated that DLAT was the most relevant gene in candidate MAGs for survival in CRC patients (P0.05). Functional clustering of DLAT co-expression genes showed that DLAT was involved in the biological process including mitotic cell cycle, prolong and termination of mitochondrial protein translation, none-coding RNA metabolism and DNA conformation change. Pathway enrichment revealed that DLAT participated in the pathways including tricarboxylic acid cycle, oxidative phosphorylation and pyruvate metabolism. DLAT co-expression molecular regulatory network was established and 10 nodes with the highest connectivity were obtained as follows: PAICS, MAD2L1, HSPD1, GART, CS, RRM1, MRPL3, BUB1, CCNB1 and CHEK1. Conclusions The expression of metabolic gene DLAT in colorectal cancer is high, but the expression is lower in advanced colorectal cancer than in early colorectal cancer, and the prognosis is poor in patients with low expression, the TNM stage is late, and lymph node metastasis is more likely to occur in patients with advanced colorectal cancer. This finding lays a foundation for the research and application of potential molecular markers related to metabolism in colorectal cancer.

Changes in expression of hippocampal α7nAChR, AChE and ChAT after sevoflurane anesthesia in neonatal rats / 中华麻醉学杂志

Objective To evaluate the changes in the expression of hippocampal α7 nicotinic ace-tylcholine receptor (α7nAChR) , acetylcholinesterase ( AChE) and choline acetyltransferase ( ChAT) after sevoflurane anesthesia in neonatal rats. Methods Seventy-two healthy Sprague-Dawley rats of both sexes, aged 7 days, weighing 25-40 g, were divided into 3 groups ( n=24 each) using a random number table method: control group ( group C) , air and oxygen group ( group A∕O) and sevoflurane group ( group S) . Rats were exposed to carrier gas ( air 1 L∕min plus oxygen 1 L∕min) for 2 h in group A∕O. Rats were ex-posed to 3. 4％ sevoflurane in carrier gas for 2 h in group S. Eight rats in each group were selected at 2 h, 1 week and 4 weeks after the end of inhalation, and sacrificed, brains were removed and hippocampal tis-sues were obtained for determination of α7nAChR, AChE and ChAT protein and mRNA by Western blot and real-time polymerase chain reaction, respectively. Results Compared with group A∕O, the expression of α7nAChR mRNA was significantly down-regulated at each time point after the end of inhalation, and the expression of TnAChR was down-regulated at 2 h after the end of inhalation and up-regulated at 1 week after the end of inhalation, the expression of AChE mRNA was up-regulated at 2 h after the end of inhalation and down-regulated at 4 weeks after the end of inhalation, the expression of AChE was down-regulated at 4 weeks after the end of inhalation, the expression of ChAT mRNA was up-regulated at 2 h after the end of in-halation, and the expression of ChAT was down-regulated at each time point after the end of inhalation in group S ( P<0. 05) . Conclusion The expression of hippocampal α7nAChR is down-regulated at first and then up-regulated after sevoflurane anesthesia, the expression of ChAT and AchE in the later period is down-regulated, the tendency of protein expression mentioned above is different from that of its mRNA ex-pression, suggesting that sevoflurane may affect the protein expression through other pathways.

Nicorandil alleviated cardiac hypoxia/reoxygenation-induced cytotoxicity via upregulating ketone body metabolism and ACAT1 activity

To study the effect of nicorandil pretreatment on ketone body metabolism and Acetyl-CoA acetyltransferase (ACAT1) activity in hypoxia/reoxygenation (H/R)-induced cardiomyocytes. In our study, we applied H9c2 cardiomyocytes cell line to evaluate the cardioprotective effects of nicorandil. We detected mitochondrial viability, cellular apoptosis, reactive oxygen species (ROS) production and calcium overloading in H9c2 cells that exposed to H/R-induced cytotoxicity. Then we evaluated whether nicorandil possibly regulated ketone body, mainly β-hydroxybutyrate (BHB) and acetoacetate (ACAC), metabolism by regulating ACAT1 and Succinyl-CoA:3-keto-acid coenzyme A transferase 1 (OXCT1) protein and gene expressions. Nicorandil protected H9c2 cardiomyocytes against H/R-induced cytotoxicity dose-dependently by mitochondria-mediated anti-apoptosis pathway. Nicorandil significantly decreased cellular apoptotic rate and enhanced the ratio of Bcl-2/Bax expressions. Further, nicorandil decreased the production of ROS and alleviated calcium overloading in H/R-induced H9c2 cells. In crucial, nicorandil upregulated ACAT1 and OXCT1 protein expressions and either of their gene expressions, contributing to increased production of cellular BHB and ACAC. Nicorandil alleviated cardiomyocytes H/R-induced cytotoxicity through upregulating ACAT1/OXCT1 activity and ketone body metabolism, which might be a potential mechanism for emerging study of nicorandil and other K(ATP) channel openers.

A pilot study of the modulation of sirtuins on arylamine -acetyltransferase 1 and 2 enzymatic activity

Arylamine -acetyltransferase (NAT; E.C. 2.3.1.5) enzymes are responsible for the biotransformation of several arylamine and hydrazine drugs by acetylation. In this process, the acetyl group transferred to the acceptor substrate produces NAT deacetylation and, in consequence, it is susceptible of degradation. Sirtuins are protein deacetylases, dependent on nicotine adenine dinucleotide, which perform post-translational modifications on cytosolic proteins. To explore possible sirtuin participation in the enzymatic activity of arylamine NATs, the expression levels of NAT1, NAT2, SIRT1 and SIRT6 in peripheral blood mononuclear cells (PBMC) from healthy subjects were examined by flow cytometry and Western blot. The activity of the sirtuins on NAT enzymatic activity was analyzed by HPLC, in the presence or absence of an agonist (resveratrol) and inhibitor (nicotinamide) of sirtuins. We detected a higher percentage of positive cells for NAT2 in comparison with NAT1, and higher numbers of SIRT1+ cells compared to SIRT6 in lymphocytes. NAT2 activity in the presence of NAM inhibitors was higher than in the presence of its substrate, but not in the presence of resveratrol. In contrast, the activity of NAT1 was not affected by sirtuins. These results showed that NAT2 activity might be modified by sirtuins.

Effects of Cornel Iridoid Glycoside on Learning-memory Ability and Brain Pathological Changes in Vascular Dementia Rats / 中国中医药信息杂志

Objective To investigate the effects of cornel iridoid glycoside (CIG) on the learning-memory ability and pathological changes in the brain of vascular dementia (VD) rats; To discuss relevant mechanism of action. Methods SD rats were randomly divided into sham-operation group, model group, positive medicine group, CIG groups low-, medium- and high-dose groups. The animal model of VD was replicated by permanent bilateral common carotid artery occlusion (2VO) in rats. Drugs were intragastrically administered 6 h after surgery and then once a day for 3 months. Morris water maze test was used to detect spatial learning-memory ability, and recognition memory was measured by the object recognition test. Immunohistochemical staining was used to detect the neuronal survival and the expression of choline acetyltransferase (ChAT) in the brain. Results Three months after permanent 2VO operation, the model rats showed a longer escape latency in Morris water maze, a lower discrimination index in the object recognition test, and a decrease in NeuN positive neuronal survival and ChAT expression in the hippocampus and cerebral cortex. Compared with the model group, intragastric administration of CIG for 3 months shortened the escape latency in Morris water maze, elevated the discrimination index in the object recognition test, and increased the NeuN positive neuronal survival in the hippocampus and cerebral cortex and ChAT expression of 2VO rats. Conclusion CIG can improve the cognitive impairment of VD model rats through protecting neurons and promoting ChAT expression.

Effects of Shenqi Xingnao Prescription on Learning and Memory Ability, Contents of Choline Acetyltransferase and Acetylcholine Esterase in Brain Tissue of Mice Models with Alzheimer Disease / 中国中医药信息杂志

Objective To observe the effects of Shenqi Xingnao Prescription on learning and memory ability, contents of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) in brain tissue in mice models with scopolamine-induced Alzheimer disease (AD); To investigate its mechanism for prevention and treatment for AD. Methods Totally 110 ICR mice were randomly divided into control group, control+Shenqi Xingnao Prescription high-dose group,model group,donepezil group,model+Shenqi Xingnao Prescription high-,medium-,and low-dose groups. The control and model group were given distilled water for gavage, and the other groups were given the corresponding medicine for gavage, once a day, for 14 days. On the 15th day, Morris water maze test and object recognition test were used to evaluate the learning and memory ability of each group. The model mice of memory impairment induced by intraperitoneal injection of scopolamine was established 20 minutes before the behavioral test. The expressions of ChAT and AChE in cortex and hippocampus were detected by Western blot. Results The results of Morris water maze test showed that compared with the control group, the model group had significant longer escape latency(P<0.05);Compared with the model group,Shenqi Xingnao Prescription medium-and high-dose groups could shorten the escape latency (P<0.05). The results of the object recognition test showed that compared with the control group, the ability of the model group to explore new things decreased and the discrimination index (DI) decreased (P<0.001);Compared with the model group,Shenqi Xingnao Prescription groups could increase the DI of model mice (P<0.05, P<0.01, P<0.001). The results of Western blot showed that the expression of AChE protein in the cortex and hippocampus of the model group was significantly higher than the control group (P<0.01); Compared with the model group, Shenqi Xingnao Prescription low- and medium-dose groups could decrease the expression of AChE in the cortex in different degrees(P<0.01);Shenqi Xingnao Prescription groups could decreaed the expression of AChE in the hippocampus (P<0.001); There was no significant statistical significance in the expression of ChAT in the cortex and hippocampus in each group.Conclusion Shenqi Xingnao Prescription can significantly improve the learning and memory ability of AD model mice induced by scopolamine, which may be related to the descent expression of AChE protein in the cortex and hippocampus of the model mice.