Gene expression analysis of cytokines and MMPs in melatonin and rhBMP-2 enhanced bone remodeling

BACKGROUND In the medical and dental fields,there is a need for studies of new therapeutic approaches for the treatment of bone defects that cause extensive bone loss.Melatonin may be an important endogenous biological factor for bone remodeling,and growth factors may enhance the repair process.AIM To evaluate the gene expression of cytokines(IL-1β,IL-6,IL-10 and TNF-α),markers of osteoclastogenesis(RANK,RANKL and OPG)and MMPs(MMP-1,MMP-2,MMP-8 and MMP-13)from the treatment of melatonin associated with an osteogenic membrane and rhBMP-2 on the recovery of a bone injury.METHODS Sixty-four rats were used and divided into 9 experimental groups and were formed according to the treatment carried out in the region of the bone lesion,which varied between the combination of 1,10 and 100μmol/L of melatonin.Gene Expression analysis was performed using real time-PCR by reading the concentration of total RNA and reverse transcription.RESULTS There were differences between groups when compared with clot or scaffold control,and improvement with a higher concentration of melatonin or rhBMP-2.The combination melatonin(1μg)with 5μg of rhBMP-2,using the guided bone regeneration technique,demonstrated some effects,albeit mild,on bone repair of critical bone defects.CONCLUSION This indicates that the approach for administering these substances needs to be reassessed,with the goal of ensuring their direct application to the affected area.Therefore,future research must be carried out,seeking to produce materials with these ideal characteristics.

Genome-Wide Identification, Evolution and Expression Analyses of GA2ox Gene Family in Brassica napus L.

Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassica napus.Here,we identified 31 GA2ox genes in B.napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes.Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm,and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons.Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups,including two C_(19)-GA2ox and two C_(20)-GA2ox clades.Group 4 is a C_(20)-GA2ox Class discovered recently.Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes.BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome.BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development,and most of them were mainly involved in abiotic responses,regulation of phytohormones and growth and development.Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons,as well as an insight into the biological functions of GA2ox family genes in B.napus.

Effects of Light and Temperature on the Expression of the Lhcb2 Gene in Pea

An approximately 800 bp cDNA ( Lhcb 2) encoding light_harvesting chlorophyll a/b_binding protein complex (type Ⅱ) was cloned from the seedling of pea ( Pisum sativum L.) with RT_PCR method. Southern blotting using special probe demonstrated that there existed one copy of Lhcb 2 in pea genome. RT_PCR and Northern blotting revealed the expression of Lhcb 2 which was regulated by light in a time_dependent expression manner. The Lhcb 2 gene didn't express untill 2 h after irradiated with white light. Low temperature (4 ℃) also affected the Lhcb 2 gene by decreasing half of its expression under 25 ℃.

Analysis of Seed-specificity of Silencing fad_2 Gene Expression in Transgenic Rapeseed Line W-4(Brassica napus L.)

This study was to investigate the efficiency and specificity of RNAi silencing on the expression of endogenous fad2 gene in transgenic line W-4. [Method] The relative expression of fad2 gene in seeds at different developmental stages of 7th, 14th, 21st and 28th day after flowering （DAF） as wel as the root, stem, leaf at winter seedling stages of both the transgenic line W-4 and non-transgenic control Westar by real-time fluorescence quantitative PCR. [Results] The results showed the relative expression of fad2 gene was gradual y increasing with the days after flowering in the seeds of the control Westar, while it was found decreasing significantly since the 21st DAF in the seeds of the line W-4. The decline was up to 60% in comparison with the control Westar. However, no significant difference in the relative expression of fad2 gene in other organs like root, stem and leaf was observed between transgenic line W-4 and non-transgenic control Westar. Fatty acid composition analysis showed the oleic acid desaturation parameter（ODP） in seeds of the line W-4 was 0.07 in average, decreased by nearly 75% than control Westar which was 0.24 in average, while no significant difference in the seedling root, stem and leaf was measured between transgenic rapeseed and control. [Conclusion] The results above validated that RNA interference in transgenic rapeseed W-4 is at a seed-specific manner, not interfering with fad2 gene expression in organs such as the root, stem and leaf. The study also found that the period of fad2 gene expres-sion decline was wel coincided with the expression of napin gene, both appeared at the 21st DAF, indicating that the expression of dsRNA of fad2 gene is precisely control ed by the napin promoter.

Identification of hub genes associated with Helicobacter pylori infection and type 2 diabetes mellitus:A pilot bioinformatics study

BACKGROUND Helicobacter pylori(H.pylori)infection is related to various extragastric diseases including type 2 diabetes mellitus(T2DM).However,the possible mechanisms connecting H.pylori infection and T2DM remain unknown.AIM To explore potential molecular connections between H.pylori infection and T2DM.METHODS We extracted gene expression arrays from three online datasets(GSE60427,GSE27411 and GSE115601).Differentially expressed genes(DEGs)commonly present in patients with H.pylori infection and T2DM were identified.Hub genes were validated using human gastric biopsy samples.Correlations between hub genes and immune cell infiltration,miRNAs,and transcription factors(TFs)were further analyzed.RESULTS A total of 67 DEGs were commonly presented in patients with H.pylori infection and T2DM.Five significantly upregulated hub genes,including TLR4,ITGAM,C5AR1,FCER1G,and FCGR2A,were finally identified,all of which are closely related to immune cell infiltration.The gene-miRNA analysis detected 13 miRNAs with at least two gene cross-links.TF-gene interaction networks showed that TLR4 was coregulated by 26 TFs,the largest number of TFs among the 5 hub genes.CONCLUSION We identified five hub genes that may have molecular connections between H.pylori infection and T2DM.This study provides new insights into the pathogenesis of H.pylori-induced onset of T2DM.

The Role of Predominant Expression of Th2 Type Cytokines Gene in the Genesis and Development of Human Gliomas

Objective: To explore the expression of Th1/Th2 cytokines gene in human gliomas and its role in the genesis and development of human gliomas.Methods: Using IL-2 and IFNγ as Th1 type cytokines, IL-4, IL-6 and IL-10 as Th2 type cytokines, the biological activity of cytokines in the supernatant of glioma cell lines was assayed by ELISA method, and the gene expression of Th1/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines were detected by RT-PCR.Results: There was predominant expression of Th2 type cytokines in human glioma cells, glioma infiltrating lymphocytes and glioma cell lines, but there was no such expression in normal brain tissues.Conclusion: It suggested that there is a relationship between the Th2 type cytokines expression in human gliomas and the immunosupressive status of human glioma patients. The predominant expression of Th2 type cytokines may play an important role in the genesis and development of human gliomas. Key words glioma - Th1/Th2 - gene expression - RT-PCR This project was supported by a grant from National Natural Sciences foundation of China (No. 30271335).

SNP Identification in α_(2A)-Adrenergic Receptor Gene in Chinese and the Effect on Gene Expression

Objective: To scan single nucleotide polymorphism ( SNP ) in Chinese alpha-2Aadrenergic receptor (α_(2A)-AR) gene and study the effects of the SNP on the gene expression.Methods: The complete sequence of α_(2A)-AR gene was analyzed with automated DNA sequencer to scanSNPs. Genomic DNA was extracted from whole blood and a 239 bp fragment containing the G/Cpolymorphism was amplified with PCR using a pair of. specific primers. PCR-RFLP was used to performthe genotyping of the SNP at the site-1 296 bp of the people in the North of China. Electrophoresismobility shift assay ( EMSA ) was used to study the binding of the 390 bp fragments (- 1 414-1 025bp) with G or C at the site-1 296 bp and nuclear extracts . Results: In our study, two SNPs werefound in α_(2A)-AR gene. Allele frequencies of the SNP at the site-1 296 bp were 0.61 and 0.39 forG and C , and the genotype frequencies were 0.34 , 0.54 and 0.13 for GG, GC and CC respectively fromthe people in the North of China. In the EMSA, a specific binding appeared in the complex ofnuclear extracts and DNA with C at-1 296 bp . Conclusion: Two SNPs exist in α_(2A)-AR gene from thepeople in the North of China , and DNA fragment with allele C of the SNP at the site-1 296 bp couldbind with a specific protein, which could influence the gene expression.

Identification and Expression Analysis of Abscisic Acid Signal Transduction Genes in Hemp Seeds

Abscisic acid(ABA)is involved in regulating diverse biological processes,but its signal transduction genes and roles in hemp seed germination are not well known.Here,the ABA signaling pathway members,PYL,PP2C and SnRK2 gene families,were identified from the hemp reference genome,including 7 CsPYL(pyrab-actin resistance1-like,ABA receptor),8 CsPP2CA(group A protein phosphatase 2c),and 7 CsSnRK2(sucrose nonfermenting1-related protein kinase 2).The content of ABA in hemp seeds in germination stage is lower than that in non-germination stage.Exogenous ABA(1 or 10μM)treatment had a significant regulatory effect on the selected PYL,PP2C,SnRK2 gene families.CsAHG3 and CsHAI1 were most significantly affected by exogenous ABA treatment.Yeast two-hybrid experiments were performed to reveal that CsPYL5,CsSnRK2.2,and CsSnRK2.3 could interact with CsPP2CA7 and demonstrate that this interaction was ABA-independent.Our results indicated that CsPYL5,CsSnRK2.2,CsSnRK2.3 and CsPP2CA7 might involve in the ABA signaling transduction pathway of hemp seeds during the hemp seed germination stages.This study suggested that novel genetic views can be brought into investigation of ABA signaling pathway in hemp seeds and lay the foundation for further exploration of the mechanism of hemp seed germination.

Effect of small interference RNA on expression of the Skp2 in human chondrocytes cell

Objective:To study the inhibitory effect of siRNA mediated by expression vectors on Skp2 expression in human chondrocytes.Method:Three Skp2 sequences siRNA-59,siRNA-318 and siRNA-504 were designed as target sites using online siRNA design tools.Skp2 siRNA expression vectors were successfully constructed in vitro by gene recombination technology,and the influence of recombinant plasmid transfection on Skp2 mRNA expression was detected.DNA electrophoresis was used to verify the results.Results:The sequence of Skp2 interference was correct by sequence analysis.The expression of Skp2 mRNA in siRNA-59,siRNA-318,siRNA-504 transfection group was significantly lower than that in no-load group and NC group(P<0.05),the inhibition rates of Skp2 mRNA in siRNA-59,siRNA-318 and siRNA-504 were respectively 60%,41%and 64%,and the siRNA-504 transfection group had the highest inhibition rate.Conclusion:The siRNA eukaryotic expression vector of Skp2 gene was constructed successfully which effectively inhibit Skp2 mRNA expression in human chondrocytes cell,and can provide strong evidence for the treatment of osteoarthritis.

Study on the effect of doxorubicin on expressions of genes encoding myocardial sarcoplasmic reticulum Ca^2＋ transport proteins and the effect of taurine on myocardial protection in rabbits

To investigate the effect of doxorubicin(DOX) on gene expression of the myocardial sarcoplasmic reticulum (SR)Ca 2+ transport proteins and the mechanism of taurine(Tau) protecting cardiac muscle cells, 9 rabbits were injected with DOX , 8 rabbits with DOX and Tau, and 9 rabbits with normal saline. Cardiac function , concentration of calcium in cardiomyocytes (Myo[Ca 2+ ] \%i\%), activity of SR Ca 2+ ATPase(SERCA2a), level of SERCA2a mRNA and Ca 2+ released channels(RYR2)mRNA were detected. The left ventricle tissues were observed by electron microscopy. The results showed that cardiac index, left ventricular systolic pressure, activity of SR Ca 2+ ATPase and level of SERCA2a mRNA decreased , while Myo[Ca 2+ ] \%i\% increased in DOX treated rabbits. DOX could not affect the level of RYR2 mRNA. Tau intervention could alleviate the increase of left ventricular diastolic pressure, Myo[Ca 2+ ] \%i\% and the decrease of SERCA2a mRNA induced by doxorubicin. The results suggested that downregulation of SERCA2a gene expression was an important mechanism of DOX induced cardiomyopathy and that Tau could partially improve the heart function by reducing calcium overload and alleviating downregulation of SERCA2a mRNA.

Expression of MTA2 Gene in Ovarian Epithelial Cancer and Its Clinical Implication

In order to investigate the roles of MTA2 in the pathogenesis of ovarian epithelial cancer, the expression of MTA2 in 4 ovarian cell lines were detected by semi-quantitative RT-PCR and Western-blot assays. MTA2 expression in normal, borderline, benign and malignant epithelial ovarian tissues was immunohistochemically examined. The expression of MTA2 mRNA and protein was detected in all of 4 cell lines of ovarian epithelial cancer. The expression of MTA2 mRNA and protein was higher in strong migration cell lines than in weak migration ones. In borderline and ma lignant ovarian tissues tested, MTA2 staining was dramatically stronger than in normal and benign tissues （P〈0. 01）. The expression levels in malignant ovarian tissues were significantly higher than that in borderline epithelial ovarian tissues （P〈0.01）. The expression of MTA2 was correlated with clinical stage, histopathological grade and lymph node metastasis. It was concluded that the high expression of MTA2 was associated with more aggressive behaviors of epithelial ovarian cancer. MTA2 provides a novel indicator of ovarian cancer.

Gene expression and MR diffusion-weighted imaging after chemoembolization in rabbit liver VX-2 tumor model

AIM: To investigate the dynamic characteristics and the correlation between PCNA, Bax, nm23, E-cadherin expression and apparent diffusion coefficient (ADC) on MR diffusion-weighted imaging (DWI) after chemoembolization in rabbit liver VX-2 tumor model. METHODS: Forty New Zealand rabbit liver VX-2 tumor models were included in the study. DWI was carried out periodically after chemoembolization. All VX-2 tumor samples in each group were examined by histopathology and Strept Avidin-Biotin Complex (SABC) immunohistochemical staining. RESULTS: The PCNA expression index in VX-2 tumors was higher than in the normal parenchyma around the tumor (P < 0.001). Nm23, Bax or E-caderin expression index in VX-2 tumors were lower than in the normal parenchyma around the tumor (all P < 0.001). PCNAand nm23 expression in the VX-2 tumor periphery first increased and then decreased (P < 0.001 and P = 0.03, respectively), while the expression of Bax and E-cadherin before and after chemoembolization was insignificant. When b-value was 100 s/mm2, there was a linear correlation between PCNA expression and ADC in the area of VX-2 tumor periphery (P < 0.001), and PCNA expression in VX-2 tumor periphery influenced the ADC. CONCLUSION: The potential of VX-2 tumor infiltrating and metastasizing decreases, while its ability to proliferate increases for a short time after chemoembolization. To some degree, the ADC value indirectly reflects the proliferation of VX-2 tumor cells.

EXPRESSION AND SWITCHING OF TH1/TH2 TYPE CYTOKINESGENE IN HUMAN GLIOMAS

Objective To study the expression and switching of Thl/Th2 cytokines gene in human gliomas and its effects on occurring and developing of human gliomas. Methods Interleukin（IL）-2 and interferon-3, represent Thl type cytokines. IL-4, IL-6, IL-10, and IL-13 represent Th2 type cytokines. The gene expressions of Th1/Th2 cytokines in human glioma cells, glioma infiltrating lymphocytes, and glioma cell lines were detected by reverse transcription polymerase chain reaction （RT-PCR）. The biological activity of cytokines in the supematant of glioma cell lines was assayed by enzyme-linked immunosorbent assay （ELISA） method. Results The total positive rates of Th1 and Th2 type cytokines gene in human glioma cells were 14.77% and 75%. The total positive rates of Th1 and Th2 type cytokines gene in glioma infiltrating lymphocytes were 22.73% and 68.17%. There was obviously predominant expression of Th2 type cytokines in human glioma tissues, glioma infiltrating lymphocytes, and glioma cell lines. There was no unbalanced expression of Th1/Th2 cytokines in normal brain tissues. Conclusion There is a predominant expression of Th2 type cytokines in human glioma cells. The switching of Th1/Th2 cytokines gene may play an important role in the occurring and developing of human gliomas.

Expression Pattern of Testis-specific Expressed Gene 2 in Cryptorchidism Model and Its Role in Apoptosis of Spermatogenic Cells

In our previous study, we identified a novel testis-specific expressed gene 2 （TSEG-2） from mouse testis. To further investigate its functions, 35 male Balb/c mice （8 weeks old） were divided into cryptorchidism group （n=20）, sham group （n=10）, and control group （n=5）. In cryptorchidism group, the right testes were anchored to the inner lateral abdominal wall. In situ hybridization （ISH） was applied to measure the localization of TSEG-2 in mouse testis. Real-time quantitative PCR was performed to detect the expression of TSEG-2 gene. Meanwhile, under the mediation of polyethylenimine （PEI）, the recombinant vector pEGFP-TSEG-2 （n=5） or empty vector （mock, n=5） was transfected into the testis of male mice. The transfection efficiencies were measured under a fluorescence microscope. The apoptosis of spermatogenic cells was detected by terminal deoxynuleotidyl-mediated nick end labeling （TUNEL）. The results showed that TSEG-2 was expressed in convoluted seminiferous tubules, more precisely, in spermatogonia and spermatocytes. As compared with sham and control groups, the TSEG-2 transcription was significantly enhanced （P〈0.05） and was correlated with apoptosis of spermatogenic cells in cryptorchid testes （P〈0.05）. PEI was efficient in mediating transfeetion of TSEG-2 into seminiferous tubules of testis. One week post-transfection, intratesticular injection of TSEG-2 resulted in increased apoptosis of spermatogenic cells in vivo （P〈0.05）. These results indicate that TSEG-2 may participate in the apoptosis of spermatogenic cells and the pathogenesis of cryptorchidism.

Expression and Chromosomal Mapping of Mouse Gpx2 Gene Encoding the Gastrointestinal Form of Glutathione Peroxidase, GPX-GI

GPX-GI is a cytosolic tetrameric Se-dependent glutathione peroxidase, similar in properties to GPX-1. Unlike the almost ubiquitous GPX-1, GPX-GI is mainly expressed in the epithelium of gastrointestinal tract. GPX-GI contributes to at least fifty percent of GPX activity in rodent small intestmal epithelium. The total GPX activity consists of at least 70% of selenium-dependent GPX activity in this compartment.By analyzing a panel of mouse mterspecies DNA from the Jackson Laboratory's backcross resource,we mapped Gpx2 gene to mouse chromosome 12 between D12Mit4 and D12Mit5, near the Ccs1 locus which contains a colon cancer susceptibility gene. A pseudogene, Gpx2-ps is mapped to mouse chromosome 7.Comparison of Gpx2 gene expression in three pairs of C57BL/6Ha and ICR/Ha mice which are respectively resistant and sensitive to dimethylhydrazine-induced colon cancer, we found a higher Gpx2 mRNA level in C57BL/6Ha colon than ICR/Ha colon. Interestingly, a lower level of GPX activity is found in the resistant strain of mice. Because GPX-1 has three times higher specific activity than GPX GI, our data suggest that the decreased GPX activity may result from a higher level of Gpx2 gene expression in those cells co-express GPx1 gene

HepG2 cells support viral replication and gene expression of hepatitis C virus genotype 4 in vitro

AIM： TO establish a cell culture system with longterm replication of hepatitis C virus （HCV） genome and expression of viral antigens in vitro. METHODS： HepG2 cell line was tested for its susceptibility to HCV by incubation with a serum from a patient with chronic hepatitis C. Cells and supernatant were harvested at various time points during the culture. Culture supernatant was tested for its ability to infect na＇ive cells. The presence of minus （antisense） RNA strand, and the detection of core and E1 antigens in cells were examined by RT-PCR and immunological techniques （flow cytometry and Western blot） respectively. RESULTS： The intracellular HCV RNA was first detected on d 3 after infection and then could be consistently detected in both cells and supernatant over a period of at least three months. The fresh cells could be infected with supernatant from cultured infected cells. Flow cytometric analysis showed surface and intracellular HCV antigen expression using in house made polyclonal antibodies （anti-core, and anti-E1）. Western blot analysis showed the expression of a cluster of immunogenic peptides at molecular weights extended between 31 and 45 kDa in an one month old culture of infected cells whereas this cluster was undetectable in uninfected HepG2 cells. CONCLUSION： HepG2 cell line is not only susceptible to HCV infection but also supports its replication in vitro. Expression of HCV structural proteins can be detected in infected HepG2 cells. These cells are also capable of shedding viral particles into culture media which in turn become infectious to uninfected cells.

HER2 gene status and the relationship with p21 protein expression in gastric cancer

Objective： We aimed to analysis the HER2 gene status and its relationship with p21 protein expression in gastric carcinoma. Methods： Fluorescence in situ hybridisation （FISH） and immunohistochemistry （IHC） techniques were used to detect HER2 gene status and p53 protein in 59 cases of gastric cancer. Results： FISH detection of HER2 gene amplification rate was 16.9% （10/59）, HER2 gene amplification in 49 cases without copy number gain and gene amplification were a total of 49.2% （29/59）. HER2 protein expression was 42.4% （25/59）, HER2 gene amplification rates in patients with ＋＋＋, ＋＋ HER2 protein expression were 3/3 and 5/8, while in patients with ＋ HER2 protein expression, it was 2/14, there was significant difference （P 0.05）. p21 protein expression rate was 49.2% （29/59）, HER2 gene amplification rates and p21 protein expression had significant difference in tumor invasion depth, lymph node metastasis （P 0.05）; had no statistical significance in histological type, age, gender differences （P 0.05）. Conclusion： HER2 gene amplification rate and gene copy number had positively correlation with p21 protein expression, HER2 gene status and expression of p21 protein combined detection can provide a reference value in gastric cancer metastasis, patient’s condition development and prognosis, it also can guide clinical development of individual treatment.

Liver expression of Nrf2-related genes in different liver diseases

BACKGROUND： The KEAP1-Nrf2 antioxidant signaling pathway is important in protecting liver from various insults. However,little is known about the expression of Nrf2-related genes in human liver in different diseases.METHODS： This study utilized normal donor liver tissues（n=35）, samples from patients with hepatocellular carcinoma（HCC, n=24）, HBV-related cirrhosis（n=27）, alcoholic cirrhosis（n=5） and end-stage liver disease（n=13）. All of the liver tissues were from the Oriental Liver Transplant Center, Beijing,China. The expressions of Nrf2 and Nrf2-related genes, including its negative regulator Kelch-like ECH-associated protein 1（KEAP1）, its targeted gene NAD（P）H-quinone oxidoreductase 1（NQO1）, glutamate-cysteine ligase catalytic subunit（GCLC） and modified subunit（GCLM）, heme oxygenase 1（HO-1） and peroxiredoxin-1（PRDX1） were evaluated. RESULTS： The expression of Nrf2 was decreased in HCC, increased in alcoholic cirrhosis and end-stage liver disease. The expression of KEAP1 was increased in all of the liver samples.The most notable finding was the increased expression of NQO1 in HCC（18-fold）, alcoholic cirrhosis（6-fold）, endstage liver disease（5-fold） and HBV-related cirrhosis（3-fold）.Peri-HCC also had 4-fold higher NQO1 m RNA as compared to the normal livers. GCLC m RNA levels were lower only in HCC, as compared to the normal livers and peri-HCC tissues.GCLM m RNA levels were higher in HBV-related cirrhosis and end-stage liver disease. HO-1 m RNA levels were increased in all liver tissues except for HCC. Peri-HCC had higher PRDX1 m RNA levels compared with HCC and normal livers.CONCLUSION： Nrf2 and Nrf2-related genes are aberrantly expressed in the liver in different diseases and the increase of NQO1 was the most notable finding, especially in HCC.

NOD2-and disease-specific gene expression profiles of peripheral blood mononuclear cells from Crohn's disease patients

AIM To investigate disease-specific gene expression profiles of peripheral blood mononuclear cells(PBMCs) from Crohn's disease(CD) patients in clinical remission.METHODS Patients with CD in clinical remission or with very low disease activity according to the Crohn's disease activity index were genotyped regarding nucleotidebinding oligomerization domain 2(NOD2),and PBMCs from wild-type(WT)-NOD2 patients,patients with homozygous or heterozygous NOD2 mutations and healthy donors were isolated for further analysis.The cells were cultured with vitamin D,peptidoglycan(PGN) and lipopolysaccharide(LPS) for defined periods of time before RNA was isolated and subjected to microarray analysis using Clariom S assays and quantitative realtime PCR.NOD2-and disease-specific gene expression profiles were evaluated with repeated measure ANOVA by a general linear model.RESULTS Employing microarray assays,a total of 267 genes were identified that were significantly up-or downregulated in PBMCs of WT-NOD2 patients,compared to healthy donors after challenge with vitamin D and/or a combination of LPS and PGN(P < 0.05;threshold:≥ 2-fold change).For further analysis by real-time PCR,genes with known impact on inflammation and immunity were selected that fulfilled predefined expression criteria.In a larger cohort of patients and controls,a disease-associated expression pattern,with higher transcript levels in vitamin D-treated PBMCs from patients,was observed for three of these genes,CLEC5 A(P < 0.030),lysozyme(LYZ;P < 0.047) and TREM1(P < 0.023).Six genes were found to be expressed in a NOD2-dependent manner(CD101,P < 0.002;CLEC5 A,P < 0.020;CXCL5,P < 0.009;IL-24,P < 0.044;ITGB2,P < 0.041;LYZ,P < 0.042).Interestingly,the highest transcript levels were observed in patients with heterozygous NOD2 mutations.CONCLUSION Our data identify CLEC5 A and LYZ as CD-and NOD2-associated genes of PBMCs and encourage further studies on their pathomechanistic roles.

Cloning and Expression Analysis of <i>RrGT2</i>Gene Related to Anthocyanin Biosynthesis in <i>Rosa rugosa</i>

At present, the research about flower color of Rosa rugosa is a very inno-vative and practical study. Glycosylation modification fulfills an important role in increasing the stability and solubility of anthocyanin in plants. In this study, based on the transcriptional database of R. rugosa, a gene with full length cDNA of 1422bp, encoding 473 amino acids, designated as RrGT2, were isolated from flowers of R. rugosa ‘Zizhi’ and then functionally characterized. According to online software prediction, the molecular formula of the protein encoded by the RrGT2 gene is C2334H3628N602O711S18, the relative molecular mass is 52,075.17 Da, and the theoretical isoelectric point is pI = 4.76. The result of the RrGT2 protein 3D model construction showed that it had the highest homology with the UDP-glycosyltransferase 74F2 protein model in the database (39.53%). Sequence alignments with the NCBI database showed that the RrGT2 protein is a member of the GTB superfamily. Homology analysis revealed that the coding regions of RrGT2 was highly specific among different species, but still had typical conserved amino acid residues called PSPG that are crucial for RrGT2 enzyme activity. RrGT2 transcripts were detected in five flowering stages and seven tissues of R. rugosa ‘Zizhi’, R. rugosa ‘Fenzizhi’ and R. rugosa ‘Baizizhi’, and their expression patterns corresponded with the accumulation of antho-cyanins. Therefore, we speculated that glycosylation of RrGT2 plays a crucial role in anthocyanin biosynthesis in R. rugosa.