喜盐鸢尾花色形成关键基因的克隆及表达分析

喜盐鸢尾(Iris halophila Pall.)及其变种蓝花喜盐鸢尾(I.halophila Pall.var.sogdiana(Bung)Grubov)因耐盐碱及其多种花色而具有盐碱地园艺开发价值。本文根据喜盐鸢尾内轮花被转录组测序结果,利用基因特异性引物从这2种植物中分别克隆了编码查尔酮合成酶(CHS)、查尔酮异构酶(CHI)、类黄酮-3',5'-羟基化酶类(F3'5'H-like)等基因的部分片段,并对它们在内轮花被中的表达水平进行实时定量PCR分析。序列分析结果确认在喜盐鸢尾中所克隆的CHS(311 bp)、CHI(457 bp)、F3'5'H-like(496 bp)3个基因(部分)未见文献报道与NCBI等数据库记录。其中F3'5'H-like基因与经典的属于细胞色素P450CYP75A亚家族的F3'5'H不同,而与万带兰的F3'5'H-like同属于CYP76AB亚家族,为一类新的蓝花相关基因。实时定量PCR表达分析结果表明,与黄花的喜盐鸢尾相比,蓝花喜盐鸢尾中CHS与F3'5'H-like显著上调表达,可能是其花色不同于喜盐鸢尾的主要原因。     

海南栽培肾茶的化学成分研究

为了解肾茶(Clerodendranthus spicatus)的化学成分,从海南栽培肾茶地上部分分离得到11个化合物,经波谱分析分别鉴定为:吐叶醇(1)、丁香脂素(2)、3,4-二羟基苯乙醇(3)、甜橙素(4)、5,6,7,4′-四甲氧基黄酮(5)、5-羟基-6,7,3′,4′-四甲氧基黄酮(6)、6-羟基-5,7,4′-三甲氧基黄酮(7)、5-羟基-6,7,3′,4′-四甲氧基黄烷酮(8)、3,3′,5-三羟基-4′,7-二甲氧基-二氢黄酮(9)、松脂素(10)和熊果酸(11)。化合物3、9和10为首次从肾茶中分离得到。对化合物1~6进行活性测试,结果表明化合物3~5对乙酰胆碱酯酶具有一定的抑制活性。     

Nucleotide sequence of 5S RNA ofMycobacterium smegmatis

³²P labelled 5S RNA isolated fromMycobacterium smegmatis was digested withT ₁ and pancreatic ribonucleases separately and fingerprinted by two dimensional high voltage electrophoresis on thin-layer DEAE-cellulose plates. The radioactive spots were sequenced and their molar yields were determined. The chain length of the 5S RNA was found to be 120. It showed resemblances to both prokaryotic and eukaryotic 5S RNAs.     

Comparison of full-length genomics sequences between dengue virus serotype 3, parental strain,and its derivatives,and B-cell epitopes prediction from envelope region

Biological markers are normally used to evaluate the candidate of live-attenuated dengue vaccines. D3V 16562 Vero 23 and D3V 16562 Vero 33 which were derivatives of D3V 16562, parental strain, showed the similar biological data. We used molecular techniques and computational tools to evaluate these derivatives. The nucleotide and amino acid sequences of the derivatives were compared to their parent. The secondary structures of untranslated regions and B-cell epitopes were predicted. The results showed that nucleotide substitutions mostly occurred in NS5 and NS5 of V2 was unusual because of amino acid change at 3349 (tryptophan →stop codon). The nucleotide substitutions in 5''UTR, prM, E, NS1, NS2A, NS3, and 3''UTR were 4, 1, 2, 2, 1, 3, and 2, respectively. The secondary structure of 5''UTR of V2 was different from P and V1. The secondary structure of 3''UTR of V2 was similar to P and certainly distinct from V1. Furthermore, B-cell epitopes prediction revealed that there were 21 epitopes of envelope and the interesting epitope was at position 297-309 because it was in domain III in which the neutralizing antibody is induced. For this study, the attenuation of derivatives was caused by the nucleotide substitutions in 5''UTR, 3''UTR, and NS5 regions. The genotypic data and B-cell epitope make the derivatives attractive for the chimeric and peptide DENV vaccine development.     

Brain 5-hydroxytryptamine level,metabolism, and binding in E1 mice

Inbred E1 mice are highly susceptible to convulsive seizures upon “throwing” stimulation. The strain is known to have an abnormal 5-hydroxytryptamine (5-HT) metabolism. In the study here 5-HT level, [¹⁴C]5-hydroxytryptophan (5-HTP) metabolism, MAO activity and [³H]5-HT receptor binding were examined in the cortex, brainstem and cerebellum. In the interictal period cortical and brainstem 5-HT level and [³H]5-HT receptor binding were significantly lower. In the same period cortical biosynthesized [¹⁴C]5-HT from [¹⁴C]5-HTP taken up was higher, and MAO activity was not changed.L-DOPA with MK486 induced a low threshold of seizures and decreased cortical 5-HT level. Abnormally functioning 5-HT neurones may exist in the E1 mouse cortex.     

RNA结合蛋白QKI-5对乳腺癌细胞MCF-7增殖的影响研究

目的:检测RNA结合蛋白QKI-5在乳腺癌细胞中的表达水平以及对癌细胞增殖能力的抑制作用。方法:通过免疫印迹实验检测QKI-5在不同乳腺癌细胞株中的表达水平,通过慢病毒感染构建能够稳定过表达QKI-5基因的细胞株,使用MTT,流式细胞仪检测细胞周期来观察过表达QKI-5对细胞增殖能力及周期的影响。结果:MCF-7细胞在三株乳腺癌细胞中QKI-5表达水平相对最低,MTT实验结果显示与对照相比,过表达QKI-5的MCF-7细胞增殖能力出现显著降低P0.05,同时细胞周期检测显示过表达QKI-5的MCF-7细胞组出现了明显的G1期阻滞,进入S期G2/M期细胞减少。结论:在乳腺癌中QKI-5的高表达可能通过抑制癌细胞周期致使细胞增殖变缓,从而导致肿瘤生长受限。     

Identification of the microRNA Expression Profile in the Regenerative Neonatal Mouse Heart by Deep Sequencing

MicroRNAs (miRNAs) are small noncoding RNAs that are involved in key biological processes, including development, differentiation, and regeneration. The global miRNA expression profile that regulates the regenerative potential of the neonatal mouse heart has not been reported. We performed deep sequencing to determine the genome-wide miRNA expression profile of the neonatal mouse heart at three key ages (1, 6, and 7 days). The miRNAs at least 1.4-fold differentially expressed between the three time points were selected for further analysis. Two miRNAs (mmu-miR-22-5p and mmu-miR-338-3p) were significantly upregulated, and nine miRNAs (mmu-miR-324-5p, mmu-miR-337-5p, mmu-miR-339-5p, mmu-miR-365-1-5p, mmu-miR-500-3p, mmu-miR-505-5p, mmu-miR-542-5p, mmu-miR-668-3p, and mmu-miR-92a-1-5p) were significantly downregulated in cardiac tissue of 7-day-old mice compared to 1- and 6-day-old mice. The expression patterns of five significantly different miRNAs were verified by quantitative real-time PCR. Furthermore, the potential targets of these putative miRNAs were suggested using miRNA target prediction tools. The candidate target genes are involved in the myocardial regenerative process, with a prominent role for the Notch signaling pathway. Our study provides a valuable resource for future investigation of the biological function of miRNAs in heart regeneration.     

肠内营养支持对克罗恩病营养障碍患者IGF-I、IGF-IR及IGFBP-5水平的影响

目的:探讨肠内营养支持对克罗恩病营养障碍患者血清IGF-I、IGF-IR及IGFBP-5水平的影响。方法:选取我院收治的克罗恩病患者92例,根据治疗方法不同分为对照组及治疗组,每组各46例。对照组予以美沙拉嗪和醋酸泼尼松龙片常规治疗,治疗组在对照组基础上加用肠内营养混悬液静脉滴注。观察并比较两组患者治疗前后血清中IGF-I、IGF-IR及IGFBP-5水平的变化情况。结果:与治疗前比较,两组患者治疗后血清IGF-I、IGF-IR及IGFBP-5水平明显降低,差异具有统计学意义(P0.05);与对照组比较,治疗组患者治疗后血清IGF-I、IGF-IR及IGFBP-5水平明显降低,差异具有统计学意义(P0.05);与对照组比较,治疗组患者治疗后的营养状况明显改善,临床总有效率明显提高,差异具有统计学意义(P0.05)。结论:肠内营养支持能够降低克罗恩病营养障碍患者血清中IGF-I、IGF-IR及IGFBP-5水平,改善肠纤维化和营养状态,提高临床疗效。     

Experimental mapping of DNA duplex shape enabled by global lineshape analyses of a nucleotide-independent nitroxide probe

Sequence-dependent variation in structure and dynamics of a DNA duplex, collectively referred to as ‘DNA shape’, critically impacts interactions between DNA and proteins. Here, a method based on the technique of site-directed spin labeling was developed to experimentally map shapes of two DNA duplexes that contain response elements of the p53 tumor suppressor. An R5a nitroxide spin label, which was covalently attached at a specific phosphate group, was scanned consecutively through the DNA duplex. X-band continuous-wave electron paramagnetic resonance spectroscopy was used to monitor rotational motions of R5a, which report on DNA structure and dynamics at the labeling site. An approach based on Pearson''s coefficient analysis was developed to collectively examine the degree of similarity among the ensemble of R5a spectra. The resulting Pearson''s coefficients were used to generate maps representing variation of R5a mobility along the DNA duplex. The R5a mobility maps were found to correlate with maps of certain DNA helical parameters, and were capable of revealing similarity and deviation in the shape of the two closely related DNA duplexes. Collectively, the R5a probe and the Pearson''s coefficient-based lineshape analysis scheme yielded a generalizable method for examining sequence-dependent DNA shapes.     

苦槛蓝叶的黄酮类成分及其荔枝霜疫霉抑制活性研究

为了解苦槛蓝(Myoporum bontioides)的化学成分,采用色谱分离法从叶中分离得到11个化合物,分别鉴定为：5,7,3’-三羟基-4’-甲氧基黄酮(1)、3,5,7,4’-四羟基-3’-甲氧基黄酮(2)、5,7,4’-三羟基-3’,5’-二甲氧基黄酮(3)、木犀草素(4)、山奈酚(5)、鼠李黄素(6)、5,7-二羟基二氢黄酮(7)、7,4’-二羟基二氢黄酮(8)、5,7,3’,4’-四羟基二氢黄酮(9)、5-O-乙酰基-3,7,3’,4’-四羟基二氢黄酮(10)和7-甲氧基香橙素(11)。除化合物4、7、11之外,其他化合物均为首次从苦槛蓝叶中分离得到。菌丝生长速率法测试表明化合物4、7～9和11对荔枝霜疫霉菌具有较好的抑菌活性。     

Further description of theLy-5 system

TheLy-5 system of alloantigens was reevaluated with the aid of an Ly-5 congenic mouse strain and an additional serological technique, the PA-SRBC rosette assay, not previously applied to Ly-5. In the PA-SRBC assay, SRBC to which PA (Staphylococcal Protein A) has been coupled react with antibodycoated cells to form rosettes. The PA-SRBC assay was more sensitive than the cytotoxicity assay in detecting Ly-5 on all cell types tested, with the exception of thymocytes, on which Ly-5 was more efficiently demonstrable by the latter test. Thus, the use of both assays gave a more complete picture of theLy-5 system. Evidently Ly-5 is expressed on most cells of the hematopoietic lineage, but on no other cells so far tested. The Ly-5⁺ cell population includes all known sets of T cells, prothymocytes, sets of B cells, cells of myeloid and monocyte-macrophage types and natural killer cells. Erythrocytes and proerythroblasts are Ly-5^?, but the Ly-5 phenotype of less differentiated erythrocytic cells is uncertain. By means of radiation chimeras, made by restoring lethally irradiated mice with Ly-5 congenic bone marrow, the weakly Ly-5⁺ phenotype of liver and of kidney was shown to be due to immigrant circulating cells. The Ly-5 phenotypes of tumors conform to this scheme of Ly-5 tissue representation. The Ly-5⁺ tumors included more than 40 leukemias tested, plasmacytomas, putative transformed equivalents of slg^?: Lyb-2⁺ early B cells, a mastocytoma, a monocytic line, and three lines related to the macrophage. The other tumors tested -a sarcoma, a spontaneous mammary carcinoma, a teratocarcinoma and a neuroblastoma — were Ly-5^?.     

G.U base pairing motifs in ribosomal RNA.

An increasing number of recognition mechanisms in RNA are found to involve G.U base pairs. In order to detect new functional sites of this type, we exhaustively analyzed the sequence alignments and secondary structures of eubacterial and chloroplast 16S and 23S rRNA, seeking positions with high levels of G.U pairs. Approximately 120 such sites were identified and classified according to their secondary structure and sequence environment. Overall biases in the distribution of G.U pairs are consistent with previously proposed structural rules: the side of the wobble pair that is subject to a loss of stacking is preferentially exposed to a secondary structure loop, where stacking is not as essential as in helical regions. However, multiple sites violate these rules and display highly conserved G.U pairs in orientations that could cause severe stacking problems. In addition, three motifs displaying a conserved G.U pair in a specific sequence/structure environment occur at an unusually high frequency. These motifs, of which two had not been reported before, involve sequences 5''UG3'' 3''GA5'' and 5''UG3'' 3''GU5'', as well as G.U pairs flanked by a bulge loop 3'' of U. The possible structures and functions of these recurrent motifs are discussed.     

Chromosome walking to the AVR1-CO39 avirulence gene of Magnaporthe grisea: discrepancy between the physical and genetic maps.

The avrCO39 gene conferring avirulence toward rice cultivar CO39 was previously mapped to chromosome 1 of Magnaporthe grisea between cosegregating markers CH5-120H and 1.2H and marker 5-10-F. In the present study, this region of the chromosome was physically mapped using RecA-mediated Achilles'' cleavage. Cleavage of genomic DNA sequences within CH5-120H and 5-10-F liberated a 610-kb restriction fragment, representing the physical distance between these markers. Chromosome walking was initiated from both markers but was curtailed due to the presence of repetitive DNA sequences and the absence of overlapping clones in cosmid libraries representing several genome equivalents. These obstacles were overcome by directly subcloning the target region after release by Achilles'' cleavage and a contig spanning avrCO39 was thus assembled. Transformation of two cosmids into a virulent recipient strain conferred a cultivar-specific avirulence phenotype thus confirming the cloning of avrCO39. Meiotic crossover points were unevenly distributed across this chromosomal region and were clustered around the avrCO39 locus. A 14-fold variation in the relationship between genetic and physical distance was measured over the avrCO39 chromosomal region. Thus the poor correlation of physical to genetic distance previously observed in M. grisea appears to be manifested over relatively short distances.     

Distribution of Young's Modulus in Porcine Corneas after Riboflavin/UVA-Induced Collagen Cross-Linking as Measured by Atomic Force Microscopy

Riboflavin/UVA-induced corneal collagen cross-linking has become an effective clinical application to treat keratoconus and other ectatic disorders of the cornea. Its beneficial effects are attributed to a marked stiffening of the unphysiologically weak stroma. Previous studies located the stiffening effect predominantly within the anterior cornea. In this study, we present an atomic force microscopy-derived analysis of the depth-dependent distribution of the Young''s modulus with a depth resolution of 5 µm in 8 cross-linked porcine corneas and 8 contralateral controls. Sagittal cryosections were fabricated from every specimen and subjected to force mapping. The mean stromal depth of the zone with effective cross-linking was found to be 219±67 µm. Within this cross-linked zone, the mean Young''s modulus declined from 49±18 kPa at the corneal surface to 46±17 kPa, 33±11 kPa, 17±5 kPa, 10±4 kPa and 10±4 kPa at stromal depth intervals of 0–50 µm, 50–100 µm, 100–150 µm, 150–200 µm and 200–250 µm, respectively. This corresponded to a stiffening by a factor of 8.1 (corneal surface), 7.6 (0–50 µm), 5.4 (50–100 µm), 3.0 (100–150 µm), 1.6 (150–200 µm), and 1.5 (200–250 µm), when compared to the Young''s modulus of the posterior 100 µm. The mean Young''s modulus within the cross-linked zone was 20±8 kPa (2.9-fold stiffening), while it was 11±4 kPa (1.7-fold stiffening) for the entire stroma. Both values were significantly distinct from the mean Young''s modulus obtained from the posterior 100 µm of the cross-linked corneas and from the contralateral controls. In conclusion, we were able to specify the depth-dependent distribution of the stiffening effect elicited by standard collagen cross-linking in porcine corneas. Apart from determining the depth of the zone with effective corneal cross-linking, we also developed a method that allows for atomic force microscopy-based measurements of gradients of Young''s modulus in soft tissues in general.     

Identification of restriction endonucleases sensitive to 5-cytosine methylation at non-CpG sites,including expanded (CAG)n/(CTG)n repeats

Most epigenetic studies assess methylation of 5'-CpG-3' sites but recent evidence indicates that non-CpG cytosine methylation occurs at high levels in humans and other species. This is most prevalent at 5'-CHG-3', where H = A, C or T, and it preferentially occurs at 5'-CpA-3' and 5'-CpT-3' sites. With the goal of facilitating the detection of non-CpG methylation, the restriction endonucleases ApeKI, BbvI, EcoP15I, Fnu4HI, MwoI and TseI were assessed for their sensitivity to 5-methylcytosine at GpCpA, GpCpT, GpCpC or GpCpG sites, where methylation is catalyzed by the DNA 5-cytosine 5'-GpC-3' methyltransferase M.CviPI. We tested a variety of sequences including various plasmid-based sites, a cloned disease-associated (CAG)83?(CTG)83 repeat and in vitro synthesized tracts of only (CAG)500?(CTG)500 or (CAG)800?(CTG)800. The repeat tracts are enriched for the preferred CpA and CpT motifs. We found that none of the tested enzymes can cleave their recognition sequences when they are 5'-GpC-3' methylated. A genomic site known to convert its non-CpG methylation levels upon C2C12 differentiation was confirmed through the use of these enzymes. These enzymes can be useful in rapidly and easily determining the most common non-CpG methylation status in various sequence contexts, as well as at expansions of (CAG)n?(CTG)n repeat tracts associated with diseases like myotonic dystrophy and Huntington disease.