Overexpression pattern,function,and clinical value of proteasome 26S subunit non-ATPase 6 in hepatocellular carcinoma

BACKGROUND In recent years,many studies have shown that proteasome 26S subunit non-ATPase 6(PSMD6)plays an important role in the occurrence and development of malignant tumours.Unfortunately,there are no reports on the evaluation of the potential role of PSMD6 in hepatocellular carcinoma(HCC).AIM To comprehensively evaluate the overexpression pattern and clinical significance of PSMD6 in HCC tissues.METHODS This study integrated PSMD6 mRNA expression profiles from 4672 HCC and 3667 non-HCC tissues,along with immunohistochemical scores from 383 HCC and adjacent tissues,to assess PSMD6 overexpression in HCC.Clustered regularly interspaced short palindromic repeats knockout technology evaluated PSMD6’s essential role in HCC cell growth.Functional enrichment analysis explored the molecular mechanism of PSMD6 abnormalities in HCC.Drug sensitivity analysis and molecular docking analysed the effect of abnormal expression of PSMD6 on the drug sensitivity of HCC cells.RESULTS The results of 41 external and two internal datasets showed that PSMD6 mRNA(SMD=0.26,95%CI:0.09-0.42,P<0.05)and protein(SMD=2.85,95%CI:1.19-4.50,P<0.05)were significantly overexpressed in HCC tissues.The integrated analysis results showed that PSMD6 had a significant overexpression pattern in HCC tissues(SMD=0.40,95%CI:0.15-0.66,P<0.05).PSMD6 knockout inhibited HCC cell growth(chronos scores<-1).Functional enrichment implicated ribosome biogenesis and RNA splicing.Significant enrichment of signalling pathways such as RNA degradation,ribosomes,and chemical carcinogenesis—reactive oxygen species.Drug sensitivity analysis and a molecular docking model showed that high expression of PSMD6 was associated with the tolerance of HCC cells to drugs such as ML323,sepantronium bromide,and GDC0810.Overexpressed PSMD6 effectively distinguished HCC tissues(AUC=0.75,95%CI:0.71-0.79).CONCLUSION This study was the first to discover that PSMD6 was overexpressed in HCC tissues.PSMD6 is essential for the growth of HCC cells and may be involved in ribosome biogenesis and RNA splicing.

Overexpression of proteasome 26S subunit non-ATPase 6 protein and its clinicopathological significance in intrahepatic cholangiocarcinoma

BACKGROUND Currently,intrahepatic cholangiocarcinoma(ICC)poses a continuing,significant health challenge,but the relationship has yet to be established between ICC and the proteasome 26S subunit non-ATPase 6(PSMD6).AIM To investigate the protein expression and clinicopathological significance of PSMD6 in ICC.METHODS The potential impact of the PSMD6 gene on the growth of ICC cell lines was analyzed using clustered regularly interspaced short palindromic repeat knockout screening technology.Forty-two paired specimens of ICC and adjacent noncancerous tissues were collected.PSMD6 protein expression was determined by immunohistochemistry.Receiver operating characteristic curve analysis was performed to validate PSMD6 expression level,and its association with ICC patients’various clinicopathological characteristics was investigated.RESULTS The PSMD6 gene was found to be essential for the growth of ICC cell lines.PSMD6 protein was significantly overexpressed in ICC tissues(P<0.001),but showed no significant association with patient age,gender,pathological grade,or tumor-node-metastasis stage(P>0.05).CONCLUSION PSMD6 can promote the growth of ICC cells,thus playing a pro-oncogenic role.

Genetic diversity of the S-type small subunit ribosomal RNA gene of Plasmodium knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia

Objective:To determine the genetic diversity of Plasmodium(P.)knowlesi isolates from Sabah,Malaysian Borneo and Peninsular Malaysia,targeting the S-type SSU rRNA gene and including aspects of natural selection and haplotype.Methods:Thirty-nine blood samples infected with P.knowlesi were collected in Sabah,Malaysian Borneo and Peninsular Malaysia.The S-type SSU rRNA gene was amplified using polymerase chain reaction,cloned into a vector,and sequenced.The natural selection and haplotype of the S-type SSU rRNA gene sequences were determined using DnaSP v6 and illustrated using NETWORK v10.This study's 39 S-type SSU rRNA sequences and eight sequences from the Genbank database were subjected to phylogenetic analysis using MEGA 11.Results:Overall,the phylogenetic analysis showed no evidence of a geographical cluster of P.knowlesi isolates from different areas in Malaysia based on the S-type SSU rRNA gene sequences.The S-type SSU rRNA gene sequences were relatively conserved and with a purifying effect.Haplotype sharing of the S-type SSU rRNA gene was observed between the P.knowlesi isolates in Sabah,Malaysian Borneo,but not between Sabah,Malaysian Borneo and Peninsular Malaysia.Conclusions:This study suggests that the S-type SSU rRNA gene of P.knowlesi isolates in Sabah,Malaysian Borneo,and Peninsular Malaysia has fewer polymorphic sites,representing the conservation of the gene.These features make the S-type SSU rRNA gene suitable for comparative studies,such as determining the evolutionary relationships and common ancestry among P.knowlesi species.

Analysis of the potential biological value of pyruvate dehydrogenase E1 subunitβin human cancer

BACKGROUND The pyruvate dehydrogenase E1 subunitβ(PDHB)gene which regulates energy metabolism is located in mitochondria.However,few studies have elucidated the role and mechanism of PDHB in different cancers.AIM To comprehensive pan-cancer analysis of PDHB was performed based on bioinformatics approaches to explore its tumor diagnostic and prognostic value and tumor immune relevance in cancer.In vitro experiments were performed to examine the biological regulation of PDHB in liver cancer.METHODS Pan-cancer data related to PDHB were obtained from the Cancer Genome Atlas(TCGA)database.Analysis of the gene expression profiles of PDHB was based on TCGA and Genotype Tissue Expression Dataset databases.Cox regression analysis and Kaplan-Meier methods were used to assess the correlation between PDHB expression and survival prognosis in cancer patients.The correlation between PDHB and receiver operating characteristic diagnostic curve,clinicopathological staging,somatic mutation,tumor mutation burden(TMB),microsatellite instability(MSI),DNA methylation,and drug susceptibility in pan-cancer was also analyzed.Various algorithms were used to analyze the correlation between PDHB and immune cell infiltration and tumor chemotaxis environment,as well as the co-expression analysis of PDHB and immune checkpoint(ICP)genes.The expression and functional phenotype of PDHB in single tumor cells were studied by single-cell sequencing,and the functional enrichment analysis of PDHB-related genes was performed.The study also validated the level of mRNA or protein expression of PDHB in several cancers.Finally,in vitro experiments verified the regulatory effect of PDHB on the proliferation,migration,and invasion of liver cancer.RESULTS PDHB was significantly and differently expressed in most cancers.PDHB was significantly associated with prognosis in patients with a wide range of cancers,including kidney renal clear cell carcinoma,kidney renal papillary cell carcinoma,breast invasive carcinoma,and brain lower grade glioma.In some cancers,PDHB expression was clearly associated with gene mutations,clinicopathological stages,and expression of TMB,MSI,and ICP genes.The expression of PDHB was closely related to the infiltration of multiple immune cells in the immune microenvironment and the regulation of tumor chemotaxis environment.In addition,single-cell sequencing results showed that PDHB correlated with different biological phenotypes of multiple cancer single cells.This study further demonstrated that down-regulation of PDHB expression inhibited the proliferation,migration,and invasion functions of hepatoma cells.CONCLUSION As a member of pan-cancer,PDHB may be a novel cancer marker with potential value in diagnosing cancer,predicting prognosis,and in targeted therapy.

Cardioprotective Potential of Cymbopogon citratus Essential Oil against Isoproterenol-induced Cardiomyocyte Hypertrophy:Possible Involvement of NLRP3 Inflammasome and Oxidative Phosphorylation Complex Subunits

Objective:Cymbopogon citratus(DC.)Stapf is a medicinal and edible herb that is widely used for the treatment of gastric,nervous and hypertensive disorders.In this study,we investigated the cardioprotective effects and mechanisms of the essential oil,the main active ingredient of Cymbopogon citratus,on isoproterenol(ISO)-induced cardiomyocyte hypertrophy.Methods:The compositions of Cymbopogon citratus essential oil(CCEO)were determined by gas chromatography-mass spectrometry.Cardiomyocytes were pretreated with 16.9µg/L CCEO for 1 h followed by 10µmol/L ISO for 24 h.Cardiac hypertrophy-related indicators and NLRP3 inflammasome expression were evaluated.Subsequently,transcriptome sequencing(RNA-seq)and target verification were used to further explore the underlying mechanism.Results:Our results showed that the CCEO mainly included citronellal(45.66%),geraniol(23.32%),and citronellol(10.37%).CCEO inhibited ISO-induced increases in cell surface area and protein content,as well as the upregulation of fetal gene expression.Moreover,CCEO inhibited ISO-induced NLRP3 inflammasome expression,as evidenced by decreased lactate dehydrogenase content and downregulated mRNA levels of NLRP3,ASC,CASP1,GSDMD,and IL-1β,as well as reduced protein levels of NLRP3,ASC,pro-caspase-1,caspase-1(p20),GSDMD-FL,GSDMD-N,and pro-IL-1β.The RNA-seq results showed that CCEO inhibited the increase in the mRNA levels of 26 oxidative phosphorylation complex subunits in ISO-treated cardiomyocytes.Our further experiments confirmed that CCEO suppressed ISO-induced upregulation of mt-Nd1,Sdhd,mt-Cytb,Uqcrq,and mt-Atp6 but had no obvious effects on mt-Col expression.Conclusion:CCEO inhibits ISO-induced cardiomyocyte hypertrophy through the suppression of NLRP3 inflammasome expression and the regulation of several oxidative phosphorylation complex subunits.

Cognitive dysfunction in schizophrenia patients caused by downregulation of γ-aminobutyric acid receptor subunits

BACKGROUND The expression pattern of gamma aminobutyric acid(GABA)receptor subunits are commonly altered in patients with schizophrenia,which may lead to nerve excitation/inhibition problems,affecting cognition,emotion,and behavior.AIM To explore GABA receptor expression and its relationship with schizophrenia and to provide insights into more effective treatments.METHODS This case-control study enrolled 126 patients with schizophrenia treated at our hospital and 126 healthy volunteers who underwent physical examinations at our hospital during the same period.The expression levels of the GABA receptor subunits were detected using 1H-magnetic resonance spectroscopy.The recognized cognitive battery tool,the MATRICS Consensus Cognitive Battery,was used to evaluate the scores for various dimensions of cognitive function.The correlation between GABA receptor subunit downregulation and schizophrenia was also analyzed.RESULTS Significant differences in GABA receptor subunit levels were found between the case and control groups(P<0.05).A significant difference was also found between the case and control groups in terms of cognitive function measures,including attention/alertness and learning ability(P<0.05).Specifically,as the expression levels of GABRA1(α1 subunit gene),GABRB2(β2 subunit gene),GABRD(δsubunit),and GABRE(εsubunit)decreased,the severity of the patients’condition increased gradually,indicating a positive correlation between the downregulation of these 4 receptor subunits and schizophrenia(P<0.05).However,the expression levels of GABRA5(α5 subunit gene)and GABRA6(α6 subunit gene)showed no significant correlation with schizophrenia(P>0.05).CONCLUSION Downregulation of the GABA receptor subunits is positively correlated with schizophrenia.In other words,when GABA receptor subunits are downregulated in patients,cognitive impairment becomes more severe.

Is 26S proteasome non-ATPase regulatory subunit 6 a potential molecular target for intrahepatic cholangiocarcinoma?

In this editorial we comment on the article by Tang et al published in the recent issue of World Journal of Hepatology.Drug therapy of intrahepatic cholangiocarcinoma(iCCA)poses an enormous challenge since only a small proportion of patients demonstrate beneficial responses to therapeutic agents.Thus,there has been a sustained search for novel molecular targets for iCCA.The study by Tang et al evaluated the role of 26S proteasome non-ATPase regulatory subunit 6(PSMD6),a 19S regulatory subunit of the proteasome,in human iCCA cells and specimens.The authors employed clustered regularly interspaced short palindromic repeat(CRISPR)knockout screening technology integrated with the computational CERES algorithm,and analyzed the human protein atlas(THPA)database and tissue microarrays.The results show that PSMD6 is a gene essential for the proliferation of 17 iCCA cell lines,and PSMD6 protein was overexpressed in iCCA tissues without a significant correlation with the clinicopathological parameters.The authors conclude that PSMD6 may play a promoting role in iCCA.The major limitations and defects of this study are the lack of detailed information of CRISPR knockout screening,in vivo experiments,and a discussion of plausible mechanistic cues,which,therefore,dampen the significance of the results.Further studies are required to verify PSMD6 as a molecular target for developing novel therapeutics for iCCA.In addition,the editorial article summarizes the latest advances in molecular targeted drugs and recently emerging immunotherapy in the clinical management of iCCA,development of proteasome inhibitors for cancer therapy,and advantages of CRISPR screening technology,computational methods,and THPA database as experimental tools for fighting cancer.We hope that these comments may provide some clues for those engaged in the field of basic and clinical research into iCCA.

Cloning and Sequence of Nicotinic Acetylcholine Receptor α Subunit from Chilo suppressalis

Nicotinic acetylcholine receptors (nAChRs) play a significant role in excitatory synaptic transmission in insects and are the target for chloronicotinyl and nereistoxin insecticides.In recent years,Chilo suppressalis,an economically important pest of rice,developed high resistance against monosultap,a nereistoxin insecticide acting on nAChR.In order to reveal the hypothesized target insensitive mechanism,studies on the molecular property of nAChR from Chilo suppressalis are required.In this study,the full length cDNA of nAChR α subunit from this pest was cloned by RT-PCR.Sequence analysis shows that it is a novel nAChR α subunit,which was named as Cs α 1(Genbank accession No.AF418987).It contains 1?997?bp nucleotides and involves an open reading frame (ORF) encoding a mature protein of 509 amino acids excluding a signal peptide of 24 amino acids.The deduced amino acid sequence was 52%-94% identical to the reported insect nAChR genes.

Identification of the Rice Vacuolar ATPase B Subunit Gene and Its Expression Pattern Analysis Under Phosphorus Deficiency

A vacuolar ATPase (V-ATPase.) B subunit gene has been cloned and characterized front a phosphorus starvation induced rice root subtractive cDNA library by suppression subtractive hybridization (SSH) method and RT-PCR amplification. This gene encodes a polypeptide of 487 amino acid residues, containing a conservative ATP binding site and with a molecular weight of 54.06 kD and an isoelectric point of 4.99, southern analysis of the. genomic DNA indicates that V-ATPase B subunit is encoded by a single gene in rice genome. The amino acid homologies of V-ATPase B subunits among different organisms range from 76% to 97% and reveals that the evolution of V-ATPase B subunit is accompanied with the biological evolution. Expression pattern analysis indicated that the maximal expression of V-ATPase B subunit gene occurred at an early stage (6 - 12 h) after phosphorus starvation in roots, and lately stage (24 - 48 It) in leaves. Under phosphorus deficiency, the up-regulated expression of V-ATPase gene was presumed to strengthen the proton transport and provide the required energy to maintain an electrochemical gradient across the tonoplast to facilitate Phosphorus transport.

Bioinformatics Analysis on α Subunit Gene of Phycobiliprotein from Spirulina maxima

[Objective]The α subunit gene of phycobiliprotein from Spirulina maxima was studied in order to provide a basis for the subsequent study of phycobiliprotein.[Method] Amino acids composition,signal peptides,hydrophobicity/hydrophilicity and trails-membrane topological structure of α subunit gene of phycobiliprotein from Spirulina maxima which registered in GenBank（GenBank AF441177） were analyzed and predicted by the tools of bioinformatic analysis.Meanwhile,phylogenetic tree was constructed based on α subunit gene of phycobiliprotein from Spirulina maxima,and its molecular evolution was also analyzed.[Result]The phycobiliprotein was rich in amino acids,which not only contained 18 kinds of essential amino acids,but also contained some non-essential amino acids like glycine,aspartic acid,etc.;Analysis on signal peptides and trails-membrane topological structure showed that the phycobiliprotein belonged to intracellular protein;Analysis on hydrophobicity/hydrophilicity showed that the phycobiliprotein belonged to hydrophilic protein;Phylogenetic analysis showed that the phycobiliprotein had a high homology with Arthrospira,which reached 99%-100%.[Conclusion]The study provided a certain reference for studying the relationship and interaction between α subunit and β subunit.

Non-SMC condensin Ⅰ complex subunit D2 and non-SMC condensin Ⅱ complex subunit D3 induces inflammation via the IKK/NF-κB pathway in ulcerative colitis

BACKGROUND Ulcerative colitis(UC)is a chronic,nonspecific intestinal inflammatory disease with undefined pathogenesis.Non-SMC condensin I complex subunit D2(NCAPD2)and non-SMC condensin II complex subunit D3(NCAPD3)play pivotal roles in chromosome assembly and segregation during both mitosis and meiosis.To date,there has been no relevant report about the functional role of NCAPD2 and NCAPD3 in UC.AIM To determine the level of NCAPD2/3 in intestinal mucosa and explore the mechanisms of NCAPD2/3 in UC.METHODS Levels of NCAPD2/3 in intestinal tissue were detected in 30 UC patients and 30 healthy individuals with in situ hybridization(ISH).In vitro,NCM60 cells were divided into the NC group,model group,si-NCAPD2 group,si-NCAPD3 group and si-NCAPD2+si-NCAPD3 group.Inflammatory cytokines were measured by ELISA,IKK and NF-κB were evaluated by western blot,and IKK nucleation and NF-κB volume were analyzed by immunofluorescence assay.RESULTS Compared with expression in healthy individuals,NCAPD2 and NCAPD3 expression in intestinal tissue was significantly upregulated(P<0.001)in UC patients.Compared with levels in the model group,IL-1β,IL-6 and TNF-αin the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups were significantly downregulated(P<0.01).IKK and NF-κB protein expression in the si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 groups was significantly decreased(P<0.01).Moreover,IKK nucleation and NF-κB volume were suppressed upon si-NCAPD2,si-NCAPD3 and si-NCAPD2+si-NCAPD3 transfection.CONCLUSION NCAPD2/3 is highly expressed in the intestinal mucosa of patients with active UC.Overexpression of NCAPD2/3 promotes the release of pro-inflammatory cytokines by modulating the IKK/NF-κB signaling pathway.

Influences of Microwave on the Cognitive Function of Chickling and the Gene Expression of NMDA Receptor Subunit

[Objective] The research aimed to study the effects of microwave on the chick embryo development and the cognitive function of chickling. [Method] The microwave which was transmitted by the permatron and was 2 450 MHz was used to simulate the microwave radiation source to radiate the hatching eggs until the chickling was hatched out. The disposable passive avoidance learning and RT-PCR were respectively used to detect the influences of microwave on the cognitive function of chickling and the expression amounts of NMDA receptor NR1 and NR2 subunits. [Result] After the microwave radiation,the avoidance rate of exposed group was significantly lower than that in the control group. Especially the avoidance rate of highest radiation intensity group was extremely significantly lower than that in the control group. Meanwhile,the body weights of two groups of chickling in the exposed group increased,and the hatching time in one group increased. Via RT-PCR analysis,the expression amount of NR2 subunit increased on the 10th day and the 15th day. The expression amount of NR1 subunit only decreased on the 15th day. [Conclusion] The microwave had the certain influence on the individual development. By changing the structure composition and function of NMDA receptor in the endbrain,the microwave made the self-regulation ability of chickling decline,which had the certain damage on the cognitive function.

Cloning and Sequence Analysis of ATPase Beta Subunit Gene from Eleutherococcus senticosus

[Objective] This study aimed to clone and analyze the cDNA of ATPase β subunit gene from Eleutherococcus senticosus.[Method] A pair of homologous primers was designed according to the chloroplast ATPase β subunit gene sequences of the known species;then the gene cDNA of E.senticosus were amplified by RT-PCR and compared with that of the known species;its structure was predicted finally.[Result] 1 099 bp of ATPase beta subunit cDNA of E.senticosus which encodes 366 amino acids was amplified by RT-PCR.Sequence comparison and structure prediction showed that amino acids encoded by the ATPase beta subunit gene of E.senticosus shared the highest homology,up to 96.41% with that of Oryza sativa.In the secondary structure,the protein contained 171 alpha helixes accounting for 46.72%,53 extended strands accounting for 14.48%,27 beta sheets accounting for 7.38% and 115 random coils which took up 31.42%.The amino acids 262-271 were the symbolic site of ATPase β subunit.The whole peptide chain had no obvious hydrophobic region and was primarily confirmed as a hydrophilic protein.[Conclusion] The cNDA of ATPase β subunit gene cloned from E.senticosus in this study will provide reference for learning the effect of energy metabolism on secondary metabolism,structure and function of ATPase in E.senticosus.

Immune Effect of Combination of Universal Molecular Adjuvant and IBDV Subunit

In order to evaluate the immune effect of the protein expressed by the universal vector pET-mLTA-CTLA-4 plus IBDV subunit, the fusion protein mLTA-CTLA-4 was expressed and purified. Protein toxicity tests were carried out on rabbits.The VP2 gene of infectious bursal virus was amplified by RT-PCR, and lately used for pET-VP2 construction. Ten-day-old free healthy chickens were chosen for a grouped test, including the mLTA-CTLA-4（at different doses） plus VP2 groups, IBDV living vaccine group and control group. Serum and mucosal samples were collected regularly and the neutralization titers of IgG and IgA were assayed, while an animal protection test was conducted to determine the protection rate. The results showed that the protein m LTA-CTLA-4 was non-toxic and its protection rate was100%. IgG or IgA levels in the IBDV vaccine group were slightly higher than those in recombinant protein groups. These results indicated that the recombinant protein mLTA-CTLA-4 could be applied with IBDV subunit vaccine to protect chickens from infection.

Cloning and Functional Analysis of Calcineurin Subunits A and B in Development and Fecundity of Nilaparvata lugens(Stål)

Serine/threonine phosphatase calcineurin(CN)is a unique but confounding calcium/calmodulin-mediated enzyme,which is composed of a catalytic subunit A(CNA)and a regulatory subunit B(CNB).We cloned six transcripts for CNA named from NlCNA-X1 to NlCNA-X6,one CNB named NlCNB1 and one CNB homologous gene NlCNBH1 from Nilaparvata lugens.All of them are constitutively transcripted in various tissues and developmental stages.The primary structure of the six isoforms showed obvious differences in the length and composition of the amino acid sequence between the two binding domains of Ca^(2+)/calmodulin(CaM)and CNB.Ca^(2+)-binding EF-hand motifs were found in NlCNB1 and NlCNBH1.The specific gene silencing of NlCNA,NlCNB1 and NlCNBH1 respectively by RNAi resulted in drastical reduction in survival rate,female weight,eclosion rate and fecundity of N.lugens.These results showed that NlCNA,NlCNB1 and NlCNBH1 were required for N.lugens growth and reproduction.The negative effects of NlCNB1 silence on nymph mortality(97%),molting malformation(90%)and female sterile(50%)were more serious than those of NlCNA or NlCNBH1.qRT-PCR and enzyme-linked immunosorbent assay(ELISA)analyses indicated that the nymphs with silenced NlCNA,NlCNB1 or NlCNBH1 showed impaired hormone and energy metabolism.In nymphs,the contents of 20-hydroxyecdysone(20E)after NlCNB1 RNAi and phenoloxidase after NlCNA RNAi were particularly decreased.These results suggested that NlCNA is involved in immunity of N.lugens by regulation of phenoloxidase,while NlCNB1 may control the growth and development of N.lugens by 20E signaling pathway in addition to interact with CNA.Injection of 70 ng/μL dsNlCNB1 resulted in 77.0%down regulation of NlCNB1,and the nymph mortality was up to 57.9%at 10 d after injection.Therefore,NlCNB1 could be a potential candidate target used for strategy design in control of N.lugens.Our results revealed the importance of CN in the regulation of the growth and development of N.lugens,which provided a basis for further study of the molecular mechanism of CN.

Characterization of High-Molecular-Weight Glutenin Subunits and Their Coding Genes from Aegilops umbellulata

The high-molecular-weight (HMW) glutenin subunits and their coding genes from Aegilops umbellulata Zhuk. (UU, 2n = 2x = 14) were characterized using SDS-PAGE analysis and molecular approaches. SDS-PAGE analysis showed that the 1Ux subunits from four different accessions possessed electrophoretic mobilities close to, or slower than, that displayed by the 1Dx2.2 subunit of common wheat. The electrophoretic mobilities of the 1Uy subunits were generally similar to those shown by the 1Dy subunits of common wheat. The complete open reading frames of the 1Ux and 1Uy genes were amplified by PCR and subsequently cloned and sequenced. Amino acid sequence comparisons suggested that the primary structure of the 1Ux and 1Uy subunits were identical to that of published HMW glutenin subunits from related species, Phylogenetic analysis indicated that the HMW glutenin subunits of Ae. umbellulata were most closely related to those encoded by the D genome of Triticeae.

人源蛋白酶体α亚基6(Proteasome subunit alpha 6)在酿酒酵母表面展示

为构建人源蛋白酶体α亚基6(α6)的酵母展示体系,研制其单克隆抗体用于抗体表位分析和研究泛素-蛋白酶体途径,建立绕过重组抗原表达及纯化制备、将展示重组抗原直接应用于抗体检测的方法.在酵母展示表达载体pICAS中引入His.tag标签,将编码α6的基因PSA6_HUMAN克隆到酵母表面展示载体pICAS-H上,用流式细胞仪检测其抗原表位活性,以表面展示α6的重组酵母细胞,结合酶联吸附免疫检测技术,建立酵母(yeast)-ELISA检测技术,应用于检测小鼠单克隆抗体及单抗效价.酵母细胞培养48h后获得抗原α6的高效表面展示,展示的α6具有良好的抗原活性和特异性,将α6的展示酵母用于yeast-ELISA的初步实验结果显示可有效检测和筛选到抗α6抗体.

Heterotrimeric G protein α subunit is involved in rice brassinosteroid response

Heterotrimeric G proteins are known to function as messengers in numerous signal transduction pathways.The nullmutation of RGA(rice heterotrimeric G protein α subunit),which encodes the α subunit of heterotrimeric G proteinin rice,causes severe dwarfism and reduced responsiveness to gibberellic acid in rice.However,less is known aboutheterotrimeric G protein in brassinosteroid(BR)signaling,one of the well-understood phytohormone pathways.In thepresent study,we used root elongation inhibition assay,lamina inclination assay and coleoptile elongation analysis todemonstrated reduced sensitivity of dl mutant plants(caused by the null mutation of RGA)to 24-epibrassinolide(24-epiBL),which belongs to brassinosteroids and plays a wide variety of roles in plant growth and development.Moreover,RGA transcript level was decreased in 24-epiBL-treated seedlings in a dose-dependent manner.Our results show thatRGA is involved in rice brassinosteroid response,which may be beneficial to elucidate the molecular mechanisms of Gprotein signaling and provide a novel perspective to understand BR signaling in higher plants.

Neuroprotective effects of autophagy inhibition on hippocampal glutamate receptor subunits after hypoxia-ischemia-induced brain damage in newborn rats

Autophagy has been suggested to participate in the pathology of hypoxic-ischemic brain damage（HIBD）.However,its regulatory role in HIBD remains unclear and was thus examined here using a rat model.To induce HIBD,the left common carotid artery was ligated in neonatal rats,and the rats were subjected to hypoxia for 2 hours.Some of these rats were intraperitoneally pretreated with the autophagy inhibitor 3-methyladenine（10 m M in 10 μL） or the autophagy stimulator rapamycin（1 g/kg） 1 hour before artery ligation.Our findings demonstrated that hypoxia-ischemia-induced hippocampal injury in neonatal rats was accompanied by increased expression levels of the autophagy-related proteins light chain 3 and Beclin-1 as well as of the AMPA receptor subunit GluR 1,but by reduced expression of GluR 2.Pretreatment with the autophagy inhibitor 3-methyladenine blocked hypoxia-ischemia-induced hippocampal injury,whereas pretreatment with the autophagy stimulator rapamycin significantly augmented hippocampal injury.Additionally,3-methyladenine pretreatment blocked the hypoxia-ischemia-induced upregulation of Glu R1 and downregulation of GluR2 in the hippocampus.By contrast,rapamycin further elevated hippocampal Glu R1 levels and exacerbated decreased GluR2 expression levels in neonates with HIBD.Our results indicate that autophagy inhibition favors the prevention of HIBD in neonatal rats,at least in part,through normalizing Glu R1 and GluR2 expression.

Oral Immunization of Mice With Vaccine of Attenuated Salmonella typhimurium Expressing Helicobacter pylori Urease B Subunit

Objective To prepare the live recombinant vaccine of attenuated Salmonella typhimurium SL3261 expressing Helicobacterpylori （H. pylori） B subunit （UreB） and to determine whether it could be used as an oral vaccine against H. pylori infection. Methods Using genomic DNA of H. pylori Sydney strain （SS1） as template, the H. pylori UreB gene fragment was amplified by PCR and subcloned into the expression vector pTC01. The recombinant plasmid pTC01-UreB was then transferred into LBS000 to obtain modified forms, and further conversed into the attenuated Salmonella typhimurium SL3261 to obtain recombinant SL3261/pCT01-UreB as an oral immunization reagent, which was then used to orally immunize Balb/c mice twice at a three-week interval. Twelve weeks later, anti-UreB IgA antibodies in intestinal fluid and IgG antibodies in sera were determined by ELISA. The relating data in control groups （including body weight, gastric inflammation, etc.） were also collected. Results The sequencing analysis showed that the UreB gene fragment amplified by PCR was consistent with the sequence of the H. pylori UreB gene. The restriction enzyme digestion revealed that the correct pTC01-UreB was obtained. SDS-PAGE and Western blot showed that a 61KD protein was expressed in SL3261/pTC01-UreB, which could be recognized by anti-H, pylori UreB antiserum and was absent in the control containing only Salmonella typhimurium SL3261 strain. The multiple oral immunization with SL3261/pTC01-UreB could significantly induce H. pylori specific mucosal IgA response as well as serum IgG responses. IFN-T and IL-10 levels were significantly increased in SL3261/pTC01-UreB group, and no obvious side effect and change in gastric inflammation were observed. Conclusion The attenuated vaccine of Salmonella typhimurium expressing H. pylori UreB can be used as an oral vaccine against H. pylori infection.