Duplicated chalcone synthase(CHS)genes modulate flavonoid production in tea plants in response to light stress

In tea plants,the abundant flavonoid compounds are responsible for the health benefits for the human body and define the astringent flavor profile.While the downstream mechanisms of flavonoid biosynthesis have been extensively studied,the role of chalcone synthase(CHS)in this secondary metabolic process in tea plants remains less clear.In this study,we compared the evolutionary profile of the flavonoid metabolism pathway and discovered that gene duplication of CHS occurred in tea plants.We identified three CsCHS genes,along with a CsCHS-like gene,as potential candidates for further functional investigation.Unlike the CsCHS-like gene,the CsCHS genes effectively restored flavonoid production in Arabidopsis chs-mutants.Additionally,CsCHS transgenic tobacco plants exhibited higher flavonoid compound accumulation compared to their wild-type counterparts.Most notably,our examination of promoter and gene expression levels for the selected CHS genes revealed distinct responses to UV-B stress in tea plants.Our findings suggest that environmental factors such as UV-B exposure could have been the key drivers behind the gene duplication events in CHS.

The gene encoding flavonol synthase contributes to lesion mimic in wheat

Lesion mimic often exhibits leaf disease-like symptoms even in the absence of pathogen infection,and is characterized by a hypersensitive-response(HR)that closely linked to plant disease resistance.Despite this,only a few lesion mimic genes have been identified in wheat.In this investigation,a lesion mimic wheat mutant named je0297 was discovered,showing no alteration in yield components when compared to the wild type(WT).Segregation ratio analysis of the F_(2)individuals resulting from the cross between the WT and the mutant revealed that the lesion mimic was governed by a single recessive gene in je0297.Using Bulked segregant analysis(BSA)and exome capture sequencing,we mapped the lesion mimic gene designated as lm6 to chromosome 6BL.Further gene fine mapping using 3315 F_(2)individuals delimited the lm6 within a 1.18 Mb region.Within this region,we identified 16 high-confidence genes,with only two displaying mutations in je0297.Notably,one of the two genes,responsible for encoding flavonol synthase,exhibited altered expression levels.Subsequent phenotype analysis of TILLING mutants confirmed that the gene encoding flavonol synthase was indeed the causal gene for lm6.Transcriptome sequencing analysis revealed that the DEGs between the WT and mutant were significantly enriched in KEGG pathways related to flavonoid biosynthesis,including flavone and flavonol biosynthesis,isoflavonoid biosynthesis,and flavonoid biosynthesis pathways.Furthermore,more than 30 pathogen infection-related(PR)genes exhibited upregulation in the mutant.Corresponding to this expression pattern,the flavonoid content in je0297 showed a significant decrease in the 4^(th)leaf,accompanied by a notable accumulation of reactive oxygen,which likely contributed to the development of lesion mimic in the mutant.This investigation enhances our comprehension of cell death signaling pathways and provides a valuable gene resource for the breeding of disease-resistant wheat.

青蒿查尔酮合成酶(AaCHS)基因家族鉴定及光调控分析

[目的]为了解青蒿(Artemisia annua L.)查尔酮合成酶(chalcone synthase,CHS)基因家族的分子进化特性及其在不同组织部位的表达情况。[方法]从Pfam数据库下载CHS蛋白HMM模型,并使用HUMMER 3.0、Blastp和CD-search鉴定出青蒿基因组中的CHS基因家族成员。采用TBtools、EXPASy、CELLO v.2.5、MEGA 7.0、MEME、SOPMA、SWISSMODEL和PlantCARE等软件对其氨基酸与碱基序列进行生物信息学分析,通过8个不同组织部位的转录组数据对AaCHS基因家族成员进行表达分析。[结果]青蒿基因组中共有16个AaCHS基因家族成员,蛋白结构分析显示AaCHS蛋白质结构并不稳定,将其分为4个组,基因结构分析显示所有AaCHS基因蛋白均具有外显子,数量为2~4,内含子数量为0~2,其中8个AaCHS基因不具有内含子。启动子顺式作用元件分析表明该基因家族成员启动子上含有大量光响应、植物激素响应元件;CD-search验证结果发现每个AaCHS蛋白均有Chal-sti-synt_N结构域。[结论]AaCHS家族成员表达分析结果表明,16个AaCHS基因在不同组织和光处理下表达不同。GO富集结果得到GO二级分类的生物过程和分子功能,生物过程共富集了80条序列;细胞组分共富集了19条序列,其中二级分类过程细胞质中富集了11条序列,数量最多,与亚细胞定位预测结果一致;推测AaCHS基因在细胞质中调控黄酮类化合物的累积。通过青蒿光调控差异表达分析,推测AaCHS7、AaCHS10在青蒿受到红光光处理中起正向调控作用。

多花黄精查尔酮合酶PcCHS的原核表达、亚细胞定位及表达分析

【目的】探究查尔酮合酶(chalcone synthase,PcCHS)基因在多花黄精类黄酮合成中的作用,为后续解析PcCHS功能以及多花黄精新品种选育提供可靠的理论依据。【方法】以多花黄精为cDNA模板,克隆多花黄精PcCHS基因的编码序列,对该基因进行生物信息学分析。通过构建PcCHS的原核表达载体,纯化目的重组蛋白,验证该酶的体外表达活性。利用瞬时超表达体系探究该基因过表达后总黄酮的含量变化。利用Gateway技术构建亚细胞定位载体35S::PcCHS-GFP,通过本氏烟草表达系统确定目的蛋白亚细胞定位情况。【结果】PcCHS基因的开放阅读框为1 251 bp,理论分子量为44.63 kD,等电点为5.89,属于亲水蛋白,与石刁柏(Asparagus officinalis)CHS亲缘关系较近。原核表达实验表明,pET28a-PcCHS经IPTG(异丙基-β-D-硫代半乳糖苷)诱导表达可溶性重组蛋白,Western-blot显示大小约为45 kD,与预期大小一致,且纯化的目的蛋白具有一定的酶活性,能催化对香豆酰辅酶A和丙二酰辅酶A转化为柚皮素查尔酮。此外,PcCHS瞬时超表达中,PcCHS组的表达量显著高于空载K组,总黄酮含量也显著高于空载K组,最高可达1.83倍。亚细胞定位结果显示,该基因在细胞膜和细胞核中发挥作用。【结论】PcCHS基因原核表达的酶具有体外酶活性,其亚细胞定位于细胞膜和细胞核,且瞬时超表达能够显著提高多花黄精叶片总黄酮含量。

Structure and expression analysis of the sucrose synthase gene family in apple

Sucrose synthases(SUS) are a family of enzymes that play pivotal roles in carbon partitioning, sink strength and plant development. A total of 11 SUS genes have been identified in the genome of Malus domestica(Md SUSs), and phylogenetic analysis revealed that the Md SUS genes were divided into three groups, named as SUS I, SUS II and SUS III, respectively. The SUS I and SUS III groups included four homologs each, whereas the SUS II group contained three homologs. SUS genes in the same group showed similar structural characteristics, such as exon number, size and length distribution. After assessing four different tissues, Md SUS1 s and Md SUS2.1 showed the highest expression in fruit, whereas Md SUS2.2/2.3 and Md SUS3 s exhibit the highest expression in shoot tips. Most Md SUSs showed decreased expression during fruit development, similar to SUS enzyme activity, but both Md SUS2.1 and Md SUS1.4 displayed opposite expression profiles. These results suggest that different Md SUS genes might play distinct roles in the sink-source sugar cycle and sugar utilization in apple sink tissues.

Polymorphism of Methionine Synthase Gene in Nuclear Families of Congenital Heart Disease

Objective To investigate the relation of methionine synthase (MS) gene variation with congenital heart disease (CHD) phenotype. Methods One hundred and ninety three CHD patients (94 males and 99 females) and their biological parents (nuclear families) in Liaoning Province were selected as the case group, and another 104 normal persons (60 males and 44 females) and their parents without family history of birth defects as the control group. For all subjects the polymorphism of MS gene A2756G locus was examined by PCR-RFLP method. Results In offspring of the control group the frequencies of MS genotype (+/ -) and allele (+) were 10.7% and 5.3%, without existence of homozygote. The MS genotype distribution and allele frequencies of CHD patients and their mothers were not significantly different from the control (P > 0.05). The frequency of allele (+) in case fathers (5.0 %) was apparently lower than that in the control (9.1%, P=0.060), and the odds ratio (OR) was 0.53 (95% CI: 0.25-1.09). There was no difference in parents' genotype combination between the two groups, and in genotype distribution among different types of CHD. Analysis of genetic transmission indicated that mutation allele (+) existed transmission disequilibrium in CHD nuclear families. The percentage of allele (+) transmitted from parents was lower than that allele (-) with OR 0.26 (95% CI: 0.11-0.60). Conclusion MS gene variation in parents is associated with occurrence of CHD in offspring, and mutation allele (+) in parents may be related with the decrease of CHD risk in offspring.

Expression of an(E)-β-farnesene synthase gene from Asian peppermint in tobacco affected aphid infestation

Aphids are major agricultural pests that cause significant yield losses in crop plants each year.(E)-β-farnesene(EβF) is the main or only component of an alarm pheromone involved in chemical communication within aphid species and particularly in the avoidance of predation. EβF also occurs in the essential oil of some plant species, and is catalyzed by EβF synthase. By using oligonucleotide primers designed from the known sequence of an EβF synthase gene from black peppermint(Mentha × piperita), two cDNA sequences, MaβFS1 and MaβFS2, were isolated from Asian peppermint(Mentha asiatica). Expression pattern analysis showed that the MaβFS1 gene exhibited higher expression in flowers than in roots, stems and leaves at the transcriptional level. Overexpression of MaβFS1 in tobacco plants resulted in emission of pure EβF ranging from 2.62 to 4.85 ng d-1g-1of fresh tissue. Tritrophic interactions involving peach aphids(Myzus persicae), and predatory lacewing(Chrysopa septempunctata) larvae demonstrated that transgenic tobacco expressing MaβFS1 had lower aphid infestation. This result suggested that the EβF synthase gene from Asian peppermint could be a good candidate for genetic engineering of agriculturally important crop plants.

Relationship Between Polymorphism of Cystathionine beta Synthase Gene and Congenital Heart Disease in Chinese Nuclear Families

Objective To study the relationship between polymorphism of cystathionine beta synthase （CBS） gene and development of congenital heart disease （CHD）. Methods One hundred and twenty-seven CHD case-parent triads were recruited from Liaoning Province as patient group, and 129 healthy subjects without family history of birth defect were simultaneously recruited as control group together with their biological parents. For all subjects the polymorphism of CBS gene G919A locus was examined by PCR-ARMS method, Results The frequencies of three genotypes （w/w, w/m, and m/m） in control group were 27.2%, 58,4%, and 14.4%, respectively, with no significant difference in gender. A significant difference in the allele frequency was found between CHD patients and controls, the wild allele frequency was 67,9% in patients and 55.7% in controls CHD parents＇ genotype distribution was significantly different from that in controls. Further comparison of each type of CHD showed that genotype frequencies in several CHD subtypes were significantly different from those in their corresponding controls. The results of TDT analysis showed that no allele transmission disequilibrium existed in CHD nuclear families. Conclusions CBS gene G919A mutation is associated with the development of CHD, and the mutated allele may decrease the risk of CHD.

Relationship Between Single Nucleotide Polymorphism of Glycogen Synthase Gene of Pacific Oyster Crassostrea gigas and Its Glycogen Content

Glycogen is important not only for the energy supplementary of oysters, but also for human consumption. High glycogen content can improve the stress survival of oyster. A key enzyme in glycogenesis is glycogen synthase that is encoded by glycogen synthase gene GYS. In this study, the relationship between single nucleotide polymorphisms(SNPs) in coding regions of Crassostrea gigas GYS(Cg-GYS) and individual glycogen content was investigated with 321 individuals from five full-sib families. Single-strand conformation polymorphism(SSCP) procedure was combined with sequencing to confirm individual SNP genotypes of Cg-GYS. Least-square analysis of variance was performed to assess the relationship of variation in glycogen content of C. gigas with single SNP genotype and SNP haplotype. As a consequence, six SNPs were found in coding regions to be significantly associated with glycogen content(P < 0.01), from which we constructed four main haplotypes due to linkage disequilibrium. Furthermore, the most effective haplotype H2(GAGGAT) had extremely significant relationship with high glycogen content(P < 0.0001). These findings revealed the potential influence of Cg-GYS polymorphism on the glycogen content and provided molecular biological information for the selective breeding of good quality traits of C. gigas.

Attenuation of Nitric Oxide Synthase Gene Expression in Rat Lung Induced by Hypoxia

In order to investigate the role of nitric oxide(NO) in pathogenesis of hypoxic pulmonary hypertension(HPH), the mean pulmonary arterial pressure(mPAP), mRNA expression of NO synthase(NOS) in lung tissues, cGMP levels, and their relationships were studied in rats exposed to hypoxia from 8 h to 28days. The results showed that mPAP began to increase in animals exposed to 10 % O2 for 8 h. Moreover, the longer the exposure, the higher the mPAP.Northern blot analysis and dot blot hybridization indicated that mRNA expression of NO in lung tissues of hypoxic rats tended to decrease with exposure days, but that of β-actin which acted as a control did not alter. The cGMP levels of plasma and lung tissues in hypoxic rats also inclined to be lower with exposure days. A marked negative correlations between the changes of cGMP levels and those of mPAP were found. It was suggested that mRNA expression of NOS gene was attenuated in hypoxic lung tissues, which may be one of important pathogenetic mechanisms of HPH.

Effects of 5-Aminolevulinic Acid on the Content of Total Flavonoids and Expression of CHS and CHI Genes in Young Apples

[ Objective] This study aimed to investigate the effects of 5-aminolevulinic acid （ALA） on the content of total flavonoids and relative expression levels of chalcoue sythase （CHS） and chalcone isomerase （CHI） genes in young apples to determine the appropriate ALA concentration and processing time. [Method] Before thinning, young apples were treated with 0 （ CK）, 100,200,300 and 400 mg/L ALA. At 12 d after ALA treatment, the content of total flavonoids in young apples was determined by ultraviolet spectrophotometry. The relative expression levels of CHS and CHI genes in young apples were determined by qPCR. [ Result ] When ALA concentration was 0 - 300 mg/L, the content of total flavonoids and relative expression levels of CHS and CHI genes in young apples were improved with the increase of ALA concentration. As ALA concentration rose to 400 rag/L, various indicators showed a downward trend. Moreover, the content of total flavouoids and relative expression levels of CHS and CHI genes in young apples treated with different concentrations of ALA were improved significantly, which reached the maximum at 9 d and declined since 12 d. [ Conclusion] Compared with CK, spraying young apples with 300 mg/L ALA at 6 -9 d before thinning was conducive to improving the content of flavanoids in thinned young apples.

胭脂萝卜RsCHS1基因克隆及表达分析

为解析RsCHS1基因在胭脂萝卜色素积累中的作用,以胭脂萝卜肉质根肉质cDNA为模板,同源克隆了RsCHS1基因的开放阅读框(ORF),采用生物信息学方法对RsCHS1蛋白的理化性质进行分析。利用实时荧光定量PCR(qRT-PCR)分析了RsCHS1基因在胭脂萝卜营养期叶、肉质根肉质和肉质根皮及花期花、荚果和肉质根肉质中的表达情况,同时构建RsCHS1基因过表达载体,异源转化拟南芥,进行RsCHS1基因功能验证。结果表明,克隆得到2个RsCHS1基因,其中RsCHS1-a基因ORF序列长765 bp,编码254个氨基酸;RsCHS1-b基因ORF序列长966 bp,编码321个氨基酸。编码蛋白二、三级结构预测显示,α-螺旋是RsCHS1主要结构元件,β-转角、片层结构、无规则卷曲分布于整个蛋白质中。qRTPCR结果表明,RsCHS1-a基因在肉质根肉质(26.8)、根皮(32.4)和花(6.7)中的相对表达量比叶(1.0)和果实(0.4)中高,RsCHS1-b基因在肉质根肉质(41.4)、根皮(41.9)和花(9.7)中的相对表达量比叶(1.0)和果实(0.6)中高,说明RsCHS1基因具有组织表达特异性。在拟南芥中过表达RsCHS1基因,发现转基因植株叶柄和茎累积大量花青素,表明RsCHS1基因在胭脂萝卜花青素积累中起到重要作用。

The Mutated Acetolactate Synthase Gene from Rice as a Non-Antibiotic Selection Marker for Transformation of Bamboo Cells

Previously, we developed a particle bombardment-mediated transformation protocol in Phyllostachys nigra bamboo by expressing hygromycin phosphotransferase gene (HPT) and neomycin phosphotransferase II gene (NPT II). Although these marker genes could introduce to several tissue cultured organs (e.g. leaves, buds, and calli) of Phyllostachs bamboo species, some organs showed a high susceptibility and/or a low selectivity to hygromycin and kanamycin. In this report, therefore, we describe advantages and technical details for generating stable transgenic bamboo cells using the particle bombardment method with the mutated-acetolactate synthase gene (mALS) from rice (W548L/S627IOsALS) as a non-antibiotic selection marker. A facile and efficient transformation was achieved with the mALS gene and enhanced fluorescent protein gene (mCherry). Approximately 490 and 1400 mCherry-expressing cells/dish/shot in average were observed in both P. bambusoides and P. nigra under fluorescent stereo-microscope. Stable transgenic bamboo cell lines were generated in a selection medium supplemented with 0.1 μM of bispyribac-sodium (BS) as ALS inhibitor. The integration of mALS gene was identified by in vivo ALS enzyme assay and a PCR-restriction fragment length polymerphism (RFLP) based detection procedures.

Gene cloning and expression analysis of limonene synthase in Syringa oblata and S.oblata var.alba

Syringa species are important ornamentals with strong floral scent,of which monoterpenes are the main component.In this study,a new monoterpene synthase gene,named SoLIM,was collected from the flowers of Syringa oblata and S.oblata var.alba using a homologous cloning method.The full-length cDNA of SoLIM was1746 bp and encoded 581 amino acids.Sequence analysis showed that SoLIM contained the DDxxD and RRx8 W motifs,which are two typical conserved monoterpene synthase motifs,and was thus classified as belonging to the Tpsb subfamily.Using quantitative reverse-transcription PCR,SoLIM was significantly expressed in the petals and pistils of S.oblata and S.oblata var.alba,respectively.SoLIM expression peaked earlier than the D-limonene emissions in the diurnal experiments,but occurred later when D-limonene had peaked during the flowering phase,indicating that differences in SoLIM gene expression and D-limonene emissions existed.The synthesis of floral scent is thus associated with diverse regulatory mechanisms that require further investigation.

Molecular cloning,characterization and expression profile of the sucrose synthase gene family in Litchi chinensis

Sucrose synthase(SUS,EC 2.4.1.13)is widely considered as a key enzyme involved in plant sucrose metabolism,and the gene family encoding different SUS isozymes has been identified and characterized in several plant species.However,to date scant information about the SUS genes is available in Litchi chinensis Sonn.Here,we identified five SUS genes in litchi.These Lc SUSs shared high levels of similarity in both nucleotide and amino acid sequences.Their gene structure,phylogenetic relationships,and expression profiles were characterized.Gene structure analysis indicated that the Lc SUSs have similar exon-intron structures.Phylogenetic analysis revealed that the five members could be classified into three groups(LcSUS1 and LcSUS2 in SUSⅡ,LcSUS4 and LcSUS5 in SUSⅢ,and LcSUS3 in SUSⅠ),demonstrating evolutionary conservation in the SUS family across litchi and other plant species.The expression levels of Lc SUSs were investigated via real-time PCR in various tissues and different developmental stages of aril.For tissues and organs,Lc SUSs exhibited distinct but partially redundant expression profiles in litchi,being predominantly expressed in young leaves(sink).During aril development,the expression pattern of LcSUS1 was consistent with the trend of sugar accumulation,indicating it may play important roles in determination of sink strength in aril.Moreover,transcript levels of LcSUS2,LcSUS4,and LcSUS5 varied between cultivars with different hexose/sucrose ratios,which may regulate the sugar composition in aril.Our results provide insights into physiological functions of SUS genes in litchi,especially roles in regulating sugar accumulation in aril.

Trehalose Synthase Gene Transfer Mediated by Agrobacterium tumefaciens Enhances Resistance to Osmotic Stress in Sugarcane

Trehalose synthase gene (TSase) from Grifola frondosa was transferred into sugarcane (Sac-charum hybrid L.) using Agrobacterium-mediated method to improve sugarcane drought-tolerance. The results indicated that embryogenic callus of sugarcane was sensitive to A. tumefaciens EHA105 strain in the transformation system employed. The high frequency PPT-resistant plants were obtained from transformated with 3 weeks callus after incubation, which reached 4.5% on average. The transgenic plants were confirmed by PCR and Southern analysis. Some transgenic plants showed multiple phenotypic alterations and some plants demonstrated improvement tolerance to osmotic stress.

EFFECT OF TNF-a AND IFN-g ON THE EXPRESSION OF INDUCIBLE NITRIC OXIDE SYNTHASE GENE AND PROLIFERATION INHIBITION OF HUMAN COLON CANCER CELL LINE

Objective: To study the expression of the inducible nitric oxide synthase (iNOS) gene and the effects of tumor necrosis factor-α(TNF-a) and interferon-γ(IFN-g)on proliferation of the continuous cultured human colon cancer cell line CCL229. Methods: Using the molecular and biochemical techniques and electron microscopy to analyze the expression of iNOS, production of NO and growth characteristics of human colon cancer cells. Results: cytokine treatment can induce expression of the iNOS gene and production of nitric oxide was significantly higher after treatment of CCL229 cells with TNF-αor IFN-γ. Treatment with either cytokine or a combination of both significantly increased levels of Malondialdehyde (MDA) over control. Furthermore, cytokine treatment increased the proliferation inhibition rate as assessed in vitro and decreased the cell proliferation index on flow cytometry. Electron microscopy showed that cells treated with cytokines had fewer pseudopodia or cell processes than control cells and that cytokine treated cells had dilatation of the mitochondria and endoplasmic reticulum and dilated vesicular or tubular cisternae. Conclusion: Our findings indicate that TNF-α and IFN-γ induce the expression of iNOS gene in CCL229 cells, which increases the production of nitric oxide, inhibits proliferation, causes lipid peroxidation, and results in ultrastructural changes.

Precise base editing of non-allelic acetolactate synthase genes confers sulfonylurea herbicide resistance in maize

Single-nucleotide polymorphisms contribute to phenotypic diversity in maize. Creation and functional annotation of point mutations has been limited by the low efficiency of conventional methods based on random mutation. An efficient tool for generating targeted single-base mutations is desirable for both functional genomics and precise genetic improvement. The objective of this study was to test the efficiency of targeted C-to-T base editing of two non-allelic acetolactate synthase(ALS) in generating sulfonylurea herbicide-resistant mutants. A CRISPR/Cas9 nickase-cytidine deaminase fused with uracil DNA glycosylase inhibitor(UGI) was employed to achieve targeted conversion of cytosine to thymine in ZmALS1 and ZmALS2. Both protoplasts and recovered mutant plants showed the activity of the cytosine base editor, with an in vivo efficiency of up to 13.8%. Transgene-free edited plants harboring a homozygous ZmALS1 mutation or a ZmALS1 and ZmALS2 double mutation were tested for their resistance at a dose of up to 15-fold the recommended limit of chlorsulfuron, a sulfonylurea herbicide widely used in agriculture. Targeted base editing of C-to-T per se and a phenotype verified in the generated mutants demonstrates the power of base editing in precise maize breeding.

Endothelial Nitric Oxide Synthase Gene Polymorphisms Associated with Susceptibility to High Altitude Pulmonary Edema in Chinese Railway Construction Workers at Qinghai-Tibet over 4500 Meters above Sea Level

Objective To examine whether the polymorphisms of endothelial nitric oxide synthase (eNOS) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Chinese railway construction workers at Qinghai-Tibet where the altitude is over 4 500 m above sea level. Methods A case-control study was conducted including 149 HAPE patients in the construction workers and 160 healthy controls randomly recruited from their co-workers, matching the patients in ethnicity, age, sex, lifestyle, and working conditions. Three polymorphisms of eNOS gene, T-786C in promoter, 894G/T in exon 7, and 27bp variable number tandem repeat (VNTR) in intron 4, were genotyped using polymerase chain reaction (PCR) and confirmed with DNA sequencing. Results The frequencies of 894T allele and heterozygous G/T of the 894G/T variant were significantly higher in HAPE patients group than in the control group (P=0.0028 and P=0.0047, respectively). However, the frequencies of the T-786C in promoter and the 27bp VNTR in intron 4 were not significantly different between the two groups. Haplotypic analysis revealed that the frequencies of two haplotypes (H3,T-T-b, b indicates 5 repeats of 27 bp VNTR; H6, C-G-a, a indicates 4 repeats of 27 bp VNTR) were significantly higher in HAPE patients (both P<0.0001). On the contrary, the frequencies of H1 (T-G-b) and H2 (T-G-a) were lower in HAPE patients than in healthy controls (both P<0.001). Conclusions Two haplotypes (T-T-b and C-G-a) may be strongly associated with susceptibility to HAPE. Compared with the individual alleles of eNOS gene, the interaction of multiple genetic markers within a haplotype may be a major determinant for the susceptibility to HAPE.

Effect of warm acupuncture on nitric oxide synthase and calcitonin gene-related peptide in a rat model of lumbar nerve root compression

BACKGROUND： Varying degrees of inflammatory responses occur during lumbar nerve root compression. Studies have shown that nitric oxide synthase （NOS） and calcitonin gene-related peptide （CGRP） are involved in secondary disc inflammation. OBJECTIVE： To observe the effects of warm acupuncture on the ultrastructure of inflammatory mediators in a rat model of lumbar nerve root compression, including NOS and CGRP contents. DESIGN, TIME AND SETTING： Randomized, controlled study, with molecular biological analysis, was performed at the Experimental Center, Sixth People＇s Hospital Affiliated to Shanghai Jiao Tong University, between September 2006 and April 2007. MATERIALS： Acupuncture needles and refined Moxa grains were purchased from Shanghai Taicheng Technology Development Co., Ltd., China; Mobic tablets were purchased from Shanghai Boehringer Ingelheim Pharmaceuticals Co., Ltd., China; enzyme linked immunosorbent assay （ELISA） kits for NOS and CGRP were purchased from ADL Biotechnology, Inc., USA. METHODS： A total of 50, healthy, adult Sprague-Dawley rats, were randomly divided into five groups normal, model, warm acupuncture, acupuncture, and drug, with 10 rats in each group. Rats in the four groups, excluding the normal group, were used to establish models of lumbar nerve root compression. After 3 days, Jiaji points were set using reinforcing-reducing manipulation in the warm acupuncture group. Moxa grains were burned on each needle, with 2 grains each daily. The acupuncture group was the same as the warm acupuncture group, with the exception of non-moxibustion. Mobic suspension （3.75 mg/kg） was used in the oral drug group, once a day. Treatment of each group lasted for 14 consecutive days. Modeling and medication were not performed in the normal group. MAIN OUTCOME MEASURES： The ultrastructure of damaged nerve roots was observed with transmission electron microscopy; NOS and CGRP contents were measured using ELISA. RESULTS： The changes of the radicular ultramicrostructure were characterized by Wallerian degeneration; nerve fibers were clearly demyelinated; axons collapsed or degenerated; outer Schwann cell cytoplasm was swollen and its nucleus was compacted. Compared with the normal group, NOS and CGRP contents in the nerve root compression zone in the model group were significantly increased （P 〈 0.01）. Nerve root edema was improved in the drug, acupuncture and the warm acupuncture groups over the model group. NOS and CGRP expressions were also decreased with the warm acupuncture group having the lowest concentration （P 〈 0.01）. CONCLUSION： In comparison to the known effects of Mobic drug and acupuncture treatments, the warm acupuncture significantly decreased NOS and CGRP expression which helped improve the ultrastructure of the compressed nerve root.