AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible i...AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.展开更多
To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas re...To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.展开更多
Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of K...Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of KB- α （IKB-α） -nuclear factor-KB （NF-KB） cascade, whereas interferon-γ （IFN-γ） acts through Janus kinase （ JAK）- signal transducer and activator of transcription 1 （ STAT1 ） signals. Heat shock factor 1 （ HSF1 ）, a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6）. But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide （NO） content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation （CHIP） was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.展开更多
Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin i...Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.展开更多
Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which mi...Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.展开更多
AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T...AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.展开更多
AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence ...AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.展开更多
Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was us...Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was used in our experiment. Twenty SD rats were randomly divided into KA 30, 60, 90, 200min and control groups. The brain sections were stained by NADPh (nicotinamide adenine dinucleotide phosphate ) diaphorase (Nd ) histochemically, and were further stained by GABA immunohistochemically.Results: Histological and immunohistochemical study revealed that in KA groups the number of Nd and GABA-positive double labelled neurons in CA3 region, CA3 region and dentate gyms was significantly reduced,compared with the control group. Conclusion: Nd coexisted with GABA in the brain. Reduction of GABA release led to relief of GABA-ergic inhibition and in the same way, reduction of NO release weakened its negative feedback modulation. Therefore neuronal synchronous paroxysmal discharges increased. GABA and NO,both having antiepileptic action, acted through different ways or different link in the same way. NO may involve in the effect of GABA-ergic neurons and play cooperative antiepileptic action with GABA.展开更多
1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-g...1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.展开更多
Isochorismate synthase(ICS) is a crucial enzyme in the salicylic acid(SA) synthesis pathway. The full-length complementary DNA(cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS...Isochorismate synthase(ICS) is a crucial enzyme in the salicylic acid(SA) synthesis pathway. The full-length complementary DNA(cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site(MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction(qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography(HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.展开更多
The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 I...The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System. It was found that NOS gene expression of cerebral cortex and caudate-putamen was markedly increased in 24 hours in ischemia group (P展开更多
Thromboxane A synthase 1 （TBXAS1） catalyses the synthesis of thromboxane A2 （TXA2）, which plays an important role in the pathogenesis of ischemic stroke. Thus, the TBXAS1 gene was investigated as a candidate gene ...Thromboxane A synthase 1 （TBXAS1） catalyses the synthesis of thromboxane A2 （TXA2）, which plays an important role in the pathogenesis of ischemic stroke. Thus, the TBXAS1 gene was investigated as a candidate gene involved in the formation of atherosclerosis. This case-control study collected peripheral blood specimens and clinical data of 370 ischemic stroke patients and 340 healthy controls in the Northern Chinese Han population from October 2010 to May 2011. Two TBXAS1 single-nucleotide polymorphisms, rs2267682 and rs10487667, were analyzed using a SNaPshot Multiplex sequencing assay to explore the relationships between the single-nucleotide polymorphisms in TBXAS1 and ischemic stroke. The TT genotype frequency and T allele frequency of rs2267682 in the patients with ischemic stroke were significantly higher than those in the controls （P 〈 0.01 and P = 0.02）. Furthermore, compared with the GG ＋ GT genotype, the TT rs2267682 genotype was associated with increased risk of ischemic stroke （odds ratio （OR） = 1.80, 95% confidence interval （CI）： 1.16–2.79, P 〈 0.01）. Multivariate logistic analysis with adjustments for confounding factors revealed that rs2267682 was still associated with ischemic stroke （OR = 1.94,95% CI ： 1.13–3.33, P = 0.02）. The frequency of the T-G haplotype in the patients was significantly higher than that in the controls according haplotype analysis （OR = 1.49, 95% CI： 1.10–2.00, P 〈 0.01）. These data reveal that the rs2267682 TBXAS1 polymorphism is associated with ischemic stroke. The TT genotype of TBXAS1 and T allele of rs2267682 increase susceptibility to ischemic stroke in this Northern Chinese Han population. The protocol has been registered with the Chinese Clinical Trial Registry （registration number： ChiCTR-COC-17013559）.展开更多
Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico clo...Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.展开更多
Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug...Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment （80 mg/day for 14.5 months） to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.展开更多
In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and developm...In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and development of Arabidopsis, we transformed Arabidopsis plants with an overexpression vector containing an aspen SUS gene (PtrSUS1). The genomic PCR confirmed the successful integration of PtrSUS1 transgene in the Arabidopsis genome. PtrSUS1 expression in transgenic Arabidopsis plants was confirmed by RT-PCR. The SUS activity was dramatically increased in all transgenic lines examined. The three selected transgenic PtrSUS1 lines exhibited faster growth and flowered about 10 days earlier compared to untransformed controls, and also possessed 133%, 139%, and 143% SUS activity compared to controls. Both fresh weights and dry biomass yields of the whole plants from these three selected transgenic lines were significantly increased to 125% of the controls. Transgenic PtrSUS1 lines also had a higher tolerance to higher concentration of sucrose which was reflective of the increased SUS activity in transgenic versus wild-type plants. The growth differences between wild-type and transgenic plants, either in root and hypocotyl length or in fresh and dry weight of whole plant, became more pronounced on the media containing higher sucrose concentrations. Taken together, these results showed that the early flowering, faster growth and increased tolerance to higher sucrose in transgenic lines were caused by the genome integration and constitutive expression of the aspen PtrSUS1 gene in transgenic Arabidopsis.展开更多
Based on known cDNAs of rice starch synthase isoforms,we constructed dsRNA interference vectors for starch synthase I(SSI)to produce transgenic plants containing starch with a moderately high amylose content.We invest...Based on known cDNAs of rice starch synthase isoforms,we constructed dsRNA interference vectors for starch synthase I(SSI)to produce transgenic plants containing starch with a moderately high amylose content.We investigated the effect of SSI suppression on grain quality traits,starch biosynthesis,and amylopectin chain distribution in rice plants exposed to two different temperature regimes.The activities and transcripts of BEs,DBEs,and other SS isoforms were further investigated to clarify the effect of SSI suppression on these key enzymes and their specific isoforms under different temperature treatments.Suppression of SSI by RNAi altered grain starch component and amylopectin chain distribution,but it exerted only a slight effect on total starch content(%)and accumulation amount(mg kernel?1)and on starch granule morphology and particle size distribution.Under normal temperature(NT),insignificant differences in kernel weight,chalky kernel proportion,chalky degree,and starch granule morphology between SSI-RNAi line and its wild type(WT)were observed.However,amylose content(AC)level and granule-bound starch synthase(GBSS)activity in rice endosperms were markedly increased by SSI-RNAi suppression.The chalky kernel proportion and chalky degree of SSIRNAi lines were significantly higher than those of WT under high temperature(HT)exposure at filling stage.Inhibition of SSI by RNAi affected amylopectin chain distribution and raised starch gelatinization temperature(GT)in two ways:directly from the SSI deficiency itself and indirectly by reducing BEIIb amounts in an SSI-deficient background.The deficiency of SSI expression led to an alteration in the susceptibility of grain chalkiness occurrence and starch gelatinization temperature to HT exposure,owing to a pleiotropic effect of SSI deficiency on the expression of other genes associated with starch biosynthesis.展开更多
Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary ce...Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.展开更多
Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins.Trehalose,a non reducing disaccharide used as protective agent and...Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins.Trehalose,a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products,can be prepared by trehalose synthase(TreS).The present work aims to construct a robust recombinant B.subtilis to achieve the secretory expression of TreS.In this study,the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B.subtilis WB800 N using pHT01 as expression vector.For avoiding the use of inducer,promoter P_(srfA)was used to replace the promoter P_(grac)in pHT01 and verify the activity of recombinant trehalose synthase.The TreS activity assay was employed to evaluate the performance of recombinant B.subtilis W800 N under different phosphate concentrations,carbon sources,carbon source concentrations,nitrogen sources and pH.The results showed that the P_(srfA)promoter had a good regulation effect under pH 8.0 condition,and the enzyme activity reached 6000 U/L.Using the PhoD as the secretory signal peptide,TreS was effectively secreted,and the extracellular enzyme activity reached 2100 U/L,accounting for 35%of the total enzyme activity.By optimizing the medium and fermentation conditions,the extracellular enzyme activity reached 6900 U/L in 5 L of fermentor,and the proportion reached 48%.The pHT01-P_(srfA)-PhoD-treS secretory recombinant B.subtilis constructed in this study has great potential in trehalose synthase production.展开更多
AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differen...AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.展开更多
Activators of sesquiterpene synthase(STS) gene expression and sesquiterpene production in Piper betle L. were examined using quantitative real time PCR and gas chromatography mass spectrometry methods, and the allelop...Activators of sesquiterpene synthase(STS) gene expression and sesquiterpene production in Piper betle L. were examined using quantitative real time PCR and gas chromatography mass spectrometry methods, and the allelopathic activity of untreated and Fusarium solani-treated betel extracts was tested on seed germination and on the shoot and root growth of Thai rice variety PSL2(Oryza sativa cv. Phitsanulok 2) and three dominant paddy weeds(Eclipta prostrata, Echinochloa crus-galli and Chloris barbata). The results demonstrated that F. solani dramatically upregulated STS expression and productions of β-cubebene, β-caryophyllene and germacrene D sesquiterpene when compared with the untreated control, and that betel extracts had a greater inhibitory effect on weeds than on rice. The effects were more clearly detected on seed germination and root growth than on shoot growth, and they were found to be dose-dependent. It is also noted that F. solani-treated extract had stronger effects than the untreated extract. The species most sensitive to the allelopathic effects was C. barbata, germination of which was completely inhibited even at a dose of 0.1 mg/mL untreated extract. With regards to rice, although betel extract at 1.0 mg/mL showed no inhibition on germination, it affected the elongation of rice roots, in addition to those of the tested weeds. The obtained data suggested that F. solani has potential as an activator of sesquiterpene allelochemical production via STS expression, the latter leading to the treated betel extract having a stronger phytotoxic effect. These results were beneficial in the promotion of natural herbicide production using biotechnology.展开更多
文摘AIM:Cydooxygenases (COX) are key enzymes for conversion of arachidonic acid to prostaglandins.Nitric oxide synthase (NOS) is the enzyme responsible for formation of nitric oxide. Both have constitutive and inducible isoforms.The inducible isoforms (iNOS and COX-2) are of great interest as regulators of tumor angiogenesis,tumorigenesis and inflammatory processes.This study was to clarify their role in pancreatic adenocarcinomas. METHODS:We investigated the immunohistochemical iNOS and COX-2 expression in 40 pancreatic ductal adenocardnomas of different grade and stage.The results were compared with microvessel density and dinicopathological data. RESULTS:Twenty-one (52.5%) of the cases showed iNOS expression,15 (37.5%) of the cases were positive for COX-2. The immunoreaction was heterogeneously distributed within the tumors.Staining intensity was different between the tumors.No correlation between iNOS and COX-2 expression was seen.There was no relationship with microvessel density. However,iNOS positive tumors developed more often distant metastases and the more malignant tumors showed a higher COX-2 expression.There was no correlation with other clinicopathological data. CONCLUSION:Approximately half of the cases expressed iNOS and COX-2.These two enzymes do not seem to be the key step in angiogenesis or carcinogenesis of pancreatic adenocarcinomas.Due to a low prevalence of COX-2 expression,chemoprevention of pancreatic carcinomas by COX-2 inhibitors can only achieve a limited success.
文摘To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.
文摘Aim Inducible nitric oxide synthase （iNOS） makes a great contribution to host defense and inflamma-tion. In many settings, lipopolysaccharide （LPS） induces iNOS expression through activation of the inhibitor of KB- α （IKB-α） -nuclear factor-KB （NF-KB） cascade, whereas interferon-γ （IFN-γ） acts through Janus kinase （ JAK）- signal transducer and activator of transcription 1 （ STAT1 ） signals. Heat shock factor 1 （ HSF1 ）, a major regulator of heat shock protein transcription, has been shown to regulate the production of pro-inflammatory cytokines such as tumor necrosis factor-α（TNF-α） and interleukin-6 （IL-6）. But it remains obscure whether and how HSF1 affects iNOS induction. Methods Western blot was used to measure the protein expression. The mRNA level was meas- ured by real time-PCR. Silence of HSF1 was achieved by small interfering RNA. Nitric oxide （NO） content and NF-KB binding activity were assayed by commercial kits. Chromatin immunoprecipitation （CHIP） was used to measure the binding activity of NF-KB and STAT1 to iNOS promoters. Results HSF1 inhibition or knockdown pre- vented the LPS- and/or IFN-γ-stimulated iNOS protein expression in cultured microglia. HSF1 inhibition blocked iNOS mRNA transcription. These inhibitory effects of HSF1 inhibition on iNOS expression were confirmed in brain tissues from endotoxemic mice. Further analysis showed that HSF1 inhibition had no effect on IKB-α degradation and NF-KB or STAT1 phosphorylation in LPS/IFN-γ-stimulated cells. The nuclear transport of active NF-KB or STAT1 was also not affected by HSF1 inhibition. But HSF1 inhibition reduced the binding of NF-KB and STAT1 to their DNA elements. In addition, HSF1 inhibition reduced NF-KB and STAT1 bindings to iNOS promoter inside the LPS/IFN-γ-stimulated cells. Conclusions This preventing effect of HSF1 inhibition on iNOS mRNA transcription presents the necessary role of HSF1 in iNOS induction.
文摘Artemisia annua L. produces small amounts of the sesquiterpenoid artemisinin, which is used for treatment of malaria. A worldwide shortage of the drug has led to intense research to increase the yield of artemisinin in the plant. In order to study the regulation of expression of a key enzyme of artemisinin biosynthesis, the promoter region of the key enzyme amorpha-4,11-diene synthase (ADS) was cloned and fused with the β-glucuronidase (GUS) reporter gene. Transgenic plants of A. annua expressing this fusion were generated and studied. Transgenic plants expressing the GUS gene were used to establish the activity of the cloned promoter by a GUS activity staining procedure. GUS under the control of the ADS promoter showed specific expression in glandular trichomes. The activity of the ADS promoter varies temporally and in old tissues essentially no GUS staining could be observed. The expression pattern of GUS and ADS in aerial parts of the transgenic plant was essentially the same indicating that the cis-elements controlling glandular trichome specific expression are included in the cloned promoter. However, some cis-element(s) that control expression in root and old leaf appears to be missing in the cloned promoter. Furthermore, qPCR was used to compare the activity of the wild-type ADS promoter with that of the cloned ADS promoter. The latter promoter showed a considerably lower activity than the wild-type promoter as judged from the levels of GUS and ADS transcripts, respectively, which may be due to the removal of an enhancing cis-element from the ADS promoter. The ADS gene is specifically expressed in stalk and secretory cells of glandular trichomes of A. annua.
基金supported by grants from the National Key Research and Development Program of China,No.2017YFA0105400(to LR)the Key Research and Development Program of Guangdong Province,No.2019B020236002(to LR)the National Natural Science Foundation of China,Nos.81972111(to LZ),81772349(to BL).
文摘Microglia,the resident monocyte of the central nervous system,play a crucial role in the response to spinal cord injury.However,the precise mechanism remains unclear.To investigate the molecular mechanisms by which microglia regulate the neuroinflammatory response to spinal cord injury,we performed single-cell RNA sequencing dataset analysis,focusing on changes in microglial subpopulations.We found that the MG1 subpopulation emerged in the acute/subacute phase of spinal cord injury and expressed genes related to cell pyroptosis,sphingomyelin metabolism,and neuroinflammation at high levels.Subsequently,we established a mouse model of contusive injury and performed intrathecal injection of siRNA and molecular inhibitors to validate the role of ceramide synthase 5 in the neuroinflammatory responses and pyroptosis after spinal cord injury.Finally,we established a PC12-BV2 cell co-culture system and found that ceramide synthase 5 and pyroptosis-associated proteins were highly expressed to induce the apoptosis of neuron cells.Inhibiting ceramide synthase 5 expression in a mouse model of spinal cord injury effectively reduced pyroptosis.Furthermore,ceramide synthase 5-induced pyroptosis was dependent on activation of the NLRP3 signaling pathway.Inhibiting ceramide synthase 5 expression in microglia in vivo reduced neuronal apoptosis and promoted recovery of neurological function.Pla2g7 formed a“bridge”between sphingolipid metabolism and ceramide synthase 5-mediated cell death by inhibiting the NLRP3 signaling pathway.Collectively,these findings suggest that inhibiting ceramide synthase 5 expression in microglia after spinal cord injury effectively suppressed microglial pyroptosis mediated by NLRP3,thereby exerting neuroprotective effects.
文摘AIML To investigate the effect and mechanism of action of the nitric oxide synthase （NOS） inhibitor NG-nitro-L-arginine methyl ester （L-NAME） on invasion and metastasis of human colorectal cancer cell line SL-174T. METHODS： Human colorectal cancer cel4 line SL-174T was cultured and treated separately with four different dosages of L-NAME for 72 h, Nitric oxide （NO） production was measured with Griess reagent, The effect of L-NAME on invasion and migration of SL-174T cells were evaluated by using Transwell chambers attached with polycarbonate filters and reconstituted basement membrane （Matrigel）, RT-PCR was performed to determine the mRNA levels of matrix metalloproteinase-2 （MMP-2） and tissue inhibitor metalloproteinase-2 （TIMP-2）,RESULTS： L-NAME could significantly inhibit NO production of SL-174T in a dose-dependent manner. After being treated for 72 h with 0.2, 0.4, 0.8, and 1.0 mmol/L L- NAME, respectively, the ability of the L-NAME treated SL- 174T cells to invade the reconstituted basement membrane decreased significantly （t = 8.056, P〈0.05; t= 14.467, P〈0.01; t= 27.785, P〈0.01; and t= 29.405, P〈0.01, respectively） and the inhibition rates were 10.29%, 19.62%, 34.08%, and 42.23%, respectively. Moreover, L-NAME could inhibit migration of SL-174T cells, and the inhibition rates were 20.76%, 24.95%, 39.43%, and 46. 85% for L-NAME at 0.2, 0.4, 0.8, and 1.0 mmol/L, respectively （t = 15.116, P〈0.01）. In addition, after treatment with L-NAME, expression of MMP-2 mRNA was significantly decreased （t = 71.238, P〈0.01） and that of TIMP-2 mRNA was markedly increased （t = -13.020, P〈0.01）. CONCLUSION： L-NAME exerts anti-invasive and anti- metastatic effects on SL-174T cell line via downregulating MNP-2 mRNA expression and upregulating TIMP-2 mRNA expression.
基金Natural Science Foudation of Hebei ProvinceEducation Department Foundation of Hebei Province.No.2002136.
文摘AIM: To study the distribution of the constitutive nitric oxide synthase (NOS) in the jejunum of adult rat. METHODS: The distribution of endothelial NOS (eNOS) was detected by immunohistochemistry. Immunofluorescence histochemical dual staining technique were used for studying the distribution of neuronal NOS (nNOS) and eNOS. The dual stained slides were observed under a confocal laser scanning microscope. RESULTS: Positive neuronal NOS (nNOS) and endothelial NOS (eNOS) cells were found to be distributed in lamina propria of villi, and the epithelial cell was not stained. eNOS was mainly located in submucosal vascular endothelia, while nNOS was mainly situated in myenteric plexus. Some cells in the villi had both nNOS and eNOS. More than 80% of the cells were positive for both nNOS and eNOS, the rest cells were positive either for nNOS or for eNOS. CONCLUSION: The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat. nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic (NANC) inhibitory nerves. eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role. eNOS and nNOS are coexpressed in some cells of lamina propria of villi. NO generated by those NOS is very important in the physiological and pathological process of small intestine.
文摘Objective: Functional significance of NO and central inhibitory neurotransmitter γ-aminobutyric(GABA) during seizures were investigated morphorlogically. Methods: A kainate-induced complex partialseizure model was used in our experiment. Twenty SD rats were randomly divided into KA 30, 60, 90, 200min and control groups. The brain sections were stained by NADPh (nicotinamide adenine dinucleotide phosphate ) diaphorase (Nd ) histochemically, and were further stained by GABA immunohistochemically.Results: Histological and immunohistochemical study revealed that in KA groups the number of Nd and GABA-positive double labelled neurons in CA3 region, CA3 region and dentate gyms was significantly reduced,compared with the control group. Conclusion: Nd coexisted with GABA in the brain. Reduction of GABA release led to relief of GABA-ergic inhibition and in the same way, reduction of NO release weakened its negative feedback modulation. Therefore neuronal synchronous paroxysmal discharges increased. GABA and NO,both having antiepileptic action, acted through different ways or different link in the same way. NO may involve in the effect of GABA-ergic neurons and play cooperative antiepileptic action with GABA.
文摘1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-D- erythritol 4-phosphate (MEP) pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.
基金supported by the National High-Tech R&D Program(863)of China(No.22011AA100605)
文摘Isochorismate synthase(ICS) is a crucial enzyme in the salicylic acid(SA) synthesis pathway. The full-length complementary DNA(cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site(MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction(qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography(HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.
文摘The effect of Radix Salviae Miltiorrhizae (RSM) on the gene expression of nitric oxide synthase (NOS) in rat brains during ischemia was studied with in situ hybridization and the results were analyzed with IBAS 2000 Image Analysis System. It was found that NOS gene expression of cerebral cortex and caudate-putamen was markedly increased in 24 hours in ischemia group (P
基金supported by a grant from the National Natural Science Foundation of China,No.81070913
文摘Thromboxane A synthase 1 （TBXAS1） catalyses the synthesis of thromboxane A2 （TXA2）, which plays an important role in the pathogenesis of ischemic stroke. Thus, the TBXAS1 gene was investigated as a candidate gene involved in the formation of atherosclerosis. This case-control study collected peripheral blood specimens and clinical data of 370 ischemic stroke patients and 340 healthy controls in the Northern Chinese Han population from October 2010 to May 2011. Two TBXAS1 single-nucleotide polymorphisms, rs2267682 and rs10487667, were analyzed using a SNaPshot Multiplex sequencing assay to explore the relationships between the single-nucleotide polymorphisms in TBXAS1 and ischemic stroke. The TT genotype frequency and T allele frequency of rs2267682 in the patients with ischemic stroke were significantly higher than those in the controls （P 〈 0.01 and P = 0.02）. Furthermore, compared with the GG ＋ GT genotype, the TT rs2267682 genotype was associated with increased risk of ischemic stroke （odds ratio （OR） = 1.80, 95% confidence interval （CI）： 1.16–2.79, P 〈 0.01）. Multivariate logistic analysis with adjustments for confounding factors revealed that rs2267682 was still associated with ischemic stroke （OR = 1.94,95% CI ： 1.13–3.33, P = 0.02）. The frequency of the T-G haplotype in the patients was significantly higher than that in the controls according haplotype analysis （OR = 1.49, 95% CI： 1.10–2.00, P 〈 0.01）. These data reveal that the rs2267682 TBXAS1 polymorphism is associated with ischemic stroke. The TT genotype of TBXAS1 and T allele of rs2267682 increase susceptibility to ischemic stroke in this Northern Chinese Han population. The protocol has been registered with the Chinese Clinical Trial Registry （registration number： ChiCTR-COC-17013559）.
文摘Resveratrol synthase (RS) is a key enzyme that plays a critical role in the resveratrol synthesis pathway. In this study, six RS genes were isolated and characterized from peanut variety “Zhenzhu Hong” by silico cloning and RT-PCR. Bioinformatics analysis showed that deduced amino acid sequences of the six cloned RS genes were highly conserved with a similarity from 95% to 99% when compared to the RS genes which had been deposited at the GenBank. The results of amino acid sequences analysis showed six RS proteins contained the Chal_Sti_Synt_N and ACP_Syn_III_C domains and can be classified to same family but with different evolutionary distance. Expression pattern analysis by QRT-PCR provided evidence indicating that the mRNA of six RS genes were primarily expressed in the peanut shell at different developmental stages with different expression levels, but only lower levels of them were evident in the peanut kernel. The subcellular localization of RS protein in onion epidermal cell was performed by Agrobacterium tumefaciens-mediated transformation and the green fluorescent was monitored by confocal fluorescence microscopy. The results indicated that, RS1 and RS5 were located in the nucleus and plasma membrane respectively, while the RS2, RS3, RS4 and RS6 were located in both nucleus inner membrane and plasma membrane. The data will provide basic information for elucidating the regulatory mechanisms and enzyme kinetics underlying the RS genes in the resveratrol synthase pathway.
基金Funding for the canine atorvastatin study was through the Alzheimer's Association IIRG-03-5673 to Elizabeth Head
文摘Alzheimer’s disease is a neurodegenerative disorder characterized by progressive cognitive impairment and neuropathology. Recent preclinical and epidemiological studies proposed statins as a possible therapeutic drug for Alzheimer’s disease, but the exact mechanisms of action are still unknown. Biliverdin reductase-A is a pleiotropic enzyme involved in cellular stress responses. It not only transforms biliverdin-IX alpha into the antioxidant bilirubin-IX alpha but its serine/threonine/ tyrosine kinase activity is able to modulate cell signaling networks. We previously reported the beneficial effects of atorvastatin treatment on biliverdin reductase-A and heme oxygenase-1 in the brains of a well characterized pre-clinical model of Alzheimer’s disease, aged beagles, together with observed improvement in cognition. Here we extend our knowledge of the effects of atorvastatin on inducible nitric oxide synthase in parietal cortex, cerebellum and liver of the same animals. We demonstrated that atorvastatin treatment （80 mg/day for 14.5 months） to aged beagles selectively increased inducible nitric oxide synthase in the parietal cortex but not in the cerebellum. In contrast, inducible nitric oxide synthase protein levels were significantly decreased in the liver. Significant positive correlations were found between biliverdin reductase-A and inducible nitric oxide synthase as well as heme oxygenase-1 protein levels in the parietal cortex. The opposite was observed in the liver. Inducible nitric oxide synthase up-regulation in the parietal cortex was positively associated with improved biliverdin reductase-A functions, whereas the oxidative-induced impairment of biliverdin reductase-A in the liver negatively affected inducible nitric oxide synthase expression, thus suggesting a role for biliverdin reductase-A in atorvastatin-dependent inducible nitric oxide synthase changes. Interestingly, increased inducible nitric oxide synthase levels in the parietal cortex were not associated with higher oxidative/nitrosative stress levels. We hypothesize that biliverdin reductase-A-dependent inducible nitric oxide synthase regulation strongly contributes to the cognitive improvement observed following atorvastatin treatment.
文摘In plants, sucrose synthase (SUS) enzymes catalyze conversion of sucrose into fructose and UDP-glucose in the presence of UDP. To investigate the impact of overexpression of heterologous SUS on the growth and development of Arabidopsis, we transformed Arabidopsis plants with an overexpression vector containing an aspen SUS gene (PtrSUS1). The genomic PCR confirmed the successful integration of PtrSUS1 transgene in the Arabidopsis genome. PtrSUS1 expression in transgenic Arabidopsis plants was confirmed by RT-PCR. The SUS activity was dramatically increased in all transgenic lines examined. The three selected transgenic PtrSUS1 lines exhibited faster growth and flowered about 10 days earlier compared to untransformed controls, and also possessed 133%, 139%, and 143% SUS activity compared to controls. Both fresh weights and dry biomass yields of the whole plants from these three selected transgenic lines were significantly increased to 125% of the controls. Transgenic PtrSUS1 lines also had a higher tolerance to higher concentration of sucrose which was reflective of the increased SUS activity in transgenic versus wild-type plants. The growth differences between wild-type and transgenic plants, either in root and hypocotyl length or in fresh and dry weight of whole plant, became more pronounced on the media containing higher sucrose concentrations. Taken together, these results showed that the early flowering, faster growth and increased tolerance to higher sucrose in transgenic lines were caused by the genome integration and constitutive expression of the aspen PtrSUS1 gene in transgenic Arabidopsis.
基金the National Key Research and Development Program of China (2017YFD0300103)the National Natural Science Foundation of China (31571602, 31871566) for its financial support to this research project
文摘Based on known cDNAs of rice starch synthase isoforms,we constructed dsRNA interference vectors for starch synthase I(SSI)to produce transgenic plants containing starch with a moderately high amylose content.We investigated the effect of SSI suppression on grain quality traits,starch biosynthesis,and amylopectin chain distribution in rice plants exposed to two different temperature regimes.The activities and transcripts of BEs,DBEs,and other SS isoforms were further investigated to clarify the effect of SSI suppression on these key enzymes and their specific isoforms under different temperature treatments.Suppression of SSI by RNAi altered grain starch component and amylopectin chain distribution,but it exerted only a slight effect on total starch content(%)and accumulation amount(mg kernel?1)and on starch granule morphology and particle size distribution.Under normal temperature(NT),insignificant differences in kernel weight,chalky kernel proportion,chalky degree,and starch granule morphology between SSI-RNAi line and its wild type(WT)were observed.However,amylose content(AC)level and granule-bound starch synthase(GBSS)activity in rice endosperms were markedly increased by SSI-RNAi suppression.The chalky kernel proportion and chalky degree of SSIRNAi lines were significantly higher than those of WT under high temperature(HT)exposure at filling stage.Inhibition of SSI by RNAi affected amylopectin chain distribution and raised starch gelatinization temperature(GT)in two ways:directly from the SSI deficiency itself and indirectly by reducing BEIIb amounts in an SSI-deficient background.The deficiency of SSI expression led to an alteration in the susceptibility of grain chalkiness occurrence and starch gelatinization temperature to HT exposure,owing to a pleiotropic effect of SSI deficiency on the expression of other genes associated with starch biosynthesis.
文摘Plant cellulose synthases (CesAs) are the key enzymes necessary for cellulose biosynthesis. In Arabidopsis, two distinct groups of three CesAs each are necessary for cellulose synthesis during primary and secondary cell wall formation. It has also been suggested that such three CesAs interact with each other to form plasma-membrane bound rosette complexes that are functional during cellulose production. However, in vivo demonstration of such assemblies of three CesAs into rosettes has not been possible. We used yeast two-hybrid assays to demonstrate the possible interactions among several CesAs from Arabidopsis and aspen via their N-terminal zinc-binding domains (ZnBDs). While strong positive interactions were detected among ZnBDs from secondary wall associated CesAs of both Arabidopsis and aspen, the intergeneric interactions between Arabidopsis and aspen CesAs were weak. Moreover, in aspen, three primary wall associated CesA ZnBDs positively interacted with each other as well as with secondary CesAs. These results suggest that ZnBDs from either primary or secondary CesAs, and even from different plant species could interact but are perhaps insufficient for specificities of such interactions among CesAs. These observations suggest that some other more specific interacting regions might exist within CesAs. It is also possible that some hitherto unknown mechanism exists in plants for assembling the rosette complexes with different compositions of CesAs. Understanding how cellulose is synthesized will have a direct impact on utilization of lignocellulosic biomass for bioenergy production.
基金Supported by the National Nature Science Foundation of China(No.31501413)Shandong key project of Research&Development plan(No.2017GSF221019)+1 种基金Young doctorate Cooperation Fund Project,Qi Lu University of Technology(Shandong Academy of Sciences)(No.2017BSHZ021)Shandong Natural Science Foundation Project(ZR2017BC072)
文摘Bacillus subtilis is a non-pathogenic Gram-positive bacterium that has been widely used to produce industrially and pharmaceutically relevant proteins.Trehalose,a non reducing disaccharide used as protective agent and additive in foodstuffs and pharmaceutical products,can be prepared by trehalose synthase(TreS).The present work aims to construct a robust recombinant B.subtilis to achieve the secretory expression of TreS.In this study,the treS gene from Pseudomonas putida ATCC47054 was amplified by PCR and further cloned and expressed in B.subtilis WB800 N using pHT01 as expression vector.For avoiding the use of inducer,promoter P_(srfA)was used to replace the promoter P_(grac)in pHT01 and verify the activity of recombinant trehalose synthase.The TreS activity assay was employed to evaluate the performance of recombinant B.subtilis W800 N under different phosphate concentrations,carbon sources,carbon source concentrations,nitrogen sources and pH.The results showed that the P_(srfA)promoter had a good regulation effect under pH 8.0 condition,and the enzyme activity reached 6000 U/L.Using the PhoD as the secretory signal peptide,TreS was effectively secreted,and the extracellular enzyme activity reached 2100 U/L,accounting for 35%of the total enzyme activity.By optimizing the medium and fermentation conditions,the extracellular enzyme activity reached 6900 U/L in 5 L of fermentor,and the proportion reached 48%.The pHT01-P_(srfA)-PhoD-treS secretory recombinant B.subtilis constructed in this study has great potential in trehalose synthase production.
文摘AIM:To investigate the effect of prednisolone,a synthetic glucocorticoid used in inflammatory diseases,on the growth of cultured osteosarcoma cells.METHODS:Two osteosarcoma cell lines with different degree of differentiation were used.SaOS2 show a rather mature phenotype,while U2 OS are negative for almost all osteoblastic markers.The cells were exposed to different concentrations of prednisolone(1-9 μmol/L) with or without antioxidants or the inhibitor of inducible nitric oxide synthase(i NOS) l-N6-(iminoethyl)-lysine-HCl(L-NIL).Cell growth was assessed by counting viable cells.The production of nitric oxide(NO) was measured in the conditioned media by the Griess method.The production of reactive oxygen species was quantified using 2'-7'-dichlorofluorescein diacetate.Western blot with specific antibodies against NOSs was performed on cell extracts.RESULTS:Prednisolone inhibited SaOS2 cell growth in a dose dependent manner.No significant effects were observed in U2OS.The inhibition of SaOS2 growth is not due to oxidative stress,because antioxidants do not rescue cell proliferation.Since high concentrations of NO inhibit bone formation,we also measured NO and found it induced in SaOS2,but not in U2 OS,exposed to prednisolone,because of the upregulation of i NOS as detected by western blot.Therefore,we treated SaOS2 with prednisolone in the presence or in the absence of L-NIL.L-NIL prevented NO release induced by prednisolone at all the concentrations apart from 9 μmol/L.At the same concentrations,we found that L-NIL rescued SaOS2 growth after exposure to prednisolone.In U2 OS cells,prednisolone did not induce NO production nor affected cell growth.All together,these data indicate that a link exists between increased amounts of NO and growth inhibition in response to prednisolone in SaOS2.CONCLUSION:Prednisolone inhibited SaOS2 proliferation by increasing the release of NO through the upregulation of i NOS,while no effect was exerted on U2OS.
基金graduate study (2016) from The National Research Council of Thailand (NRCT) for financial support
文摘Activators of sesquiterpene synthase(STS) gene expression and sesquiterpene production in Piper betle L. were examined using quantitative real time PCR and gas chromatography mass spectrometry methods, and the allelopathic activity of untreated and Fusarium solani-treated betel extracts was tested on seed germination and on the shoot and root growth of Thai rice variety PSL2(Oryza sativa cv. Phitsanulok 2) and three dominant paddy weeds(Eclipta prostrata, Echinochloa crus-galli and Chloris barbata). The results demonstrated that F. solani dramatically upregulated STS expression and productions of β-cubebene, β-caryophyllene and germacrene D sesquiterpene when compared with the untreated control, and that betel extracts had a greater inhibitory effect on weeds than on rice. The effects were more clearly detected on seed germination and root growth than on shoot growth, and they were found to be dose-dependent. It is also noted that F. solani-treated extract had stronger effects than the untreated extract. The species most sensitive to the allelopathic effects was C. barbata, germination of which was completely inhibited even at a dose of 0.1 mg/mL untreated extract. With regards to rice, although betel extract at 1.0 mg/mL showed no inhibition on germination, it affected the elongation of rice roots, in addition to those of the tested weeds. The obtained data suggested that F. solani has potential as an activator of sesquiterpene allelochemical production via STS expression, the latter leading to the treated betel extract having a stronger phytotoxic effect. These results were beneficial in the promotion of natural herbicide production using biotechnology.
