Structural properties of DNA oligomers containing (GACX)(n) and (GAXC)(n) tandem repeats

The antisense DNA sequence of mature mouse micro RNA, miR341, includes three repeats of the tetranucleotide (GACC). The -GAC- repeat is known to form a parallel duplex, in acidic environments. The thermal melting profile of miR341 DNA, at pH 4, 5, and 6 indicates the formation of a very stable structure, which loses its stability when pH is increased. Thus, the addition of a cytosine at the 3' end of the (GAC) motif preserves the molecule's potential to fold into an unusual structure at low pH. The effect of modifying the nucleotide composition of the GACC sequence on the secondary structures formed by oligomers containing seven tandem repeats of the altered motifs was examined here. UV melting profiles were determined, as a function of pH, for 28-mers of the two series (GAXC)(7) and (GACX)(7) (X= A/C/T/G)(.) The sequence (GACC)(7) was found to be extremely sensitive to pH variations, with a stable structure formed at pH 5 (T(m) ≥ 60°C). NMR spectroscopy established that the low pH structure is not B-DNA. (GACA)(7) and (GACT)(7) also formed stable structures at low pH but the addition of guanine at the 3'end, as seen in the (GACG) series resulted in the loss of this property. Introducing a break in the 5'-GAC-3' motif, explored in the (GAXC) series, also inhibits formation of stable structures under acidic conditions.     

酿酒酵母菌核糖体RNA沉降系数的初步研究

为研究酿酒酵母菌核糖体RNA(rRNA)的沉降系数,用酶解法和液氮研磨法裂解酿酒酵母菌的细胞壁,Trizol Reagent提取其总RNA,同时提取小白鼠和斑马鱼的总RNA进行比较.经紫外分光光度计检测和甲醛琼脂糖变性胶电泳后,RNA纯度好,条带清晰,无弥散或降解现象.试验发现,与酶解法相比,用液氮研磨法破碎酿酒酵母菌细胞壁提取总RNA所用的成本低,时间少,产率和纯度高,适用于少量样品RNA的提取.同时,酿酒酵母菌与斑马鱼和小白鼠总RNA电泳图谱表明,三者的"18S rRNA"在条带大小方面差异较小,而"28S rRNA"差异较大.利用分析型离心机测得的酿酒酵母菌两个较大rRNA的沉降系数分别为24.7S和18.1S.研究结果表明了真核生物rRNA种类的多样性.     

Identification of a competitive translation determinant in the 3' untranslated region of alfalfa mosaic virus coat protein mRNA.

We report that the competitive translational activity of alfalfa mosaic virus coat protein mRNA (CP RNA), a nonadenylated mRNA, is determined in part by the 3' untranslated region (UTR). Competitive translation was characterized both in vitro, with cotranslation assays, and in vivo, with microinjected Xenopus laevis oocytes. In wheat germ extracts, coat protein synthesis was constant when a fixed amount of full-length CP RNA was cotranslated with increasing concentrations of competitor globin mRNA. However, translation of CP RNA lacking the 3' UTR decreased significantly under competitive conditions. RNA stabilities were equivalent. In X. laevis oocytes, which are translationally saturated and are an inherently competitive translational environment, full-length CP RNA assembled into large polysomes and coat protein synthesis was readily detectable. Alternatively, CP RNA lacking the 3' UTR sedimented as small polysomes, and little coat protein was detected. Again, RNA stabilities were equivalent. Site-directed mutagenesis was used to localize RNA sequences or structures required for competitive translation. Since the CP RNA 3' UTR has an unusually large number of AUG nucleotide triplets, two AUG-containing sites were altered in full-length RNA prior to oocyte injections. Nucleotide substitutions at the sequence GAUG, 20 nucleotides downstream of the coat protein termination codon, specifically reduced full-length CP RNA translation, while similar substitutions at the next AUG triplet had little effect on translation. The competitive influence of the 3' UTR could be explained by RNA-protein interactions that affect translation initiation or by ribosome reinitiation at downstream AUG codons, which would increase the number of ribosomes committed to coat protein synthesis.     

Isolation and characterization of nisin-producing Lactococcus lactis subsp. lactis from bean-sprouts

Bacterial isolates from bean-sprouts were screened for anti- Listeria monocytogenes bacteriocins using a well diffusion method. Thirty-four of 72 isolates inhibited the growth of L.monocytogenes Scott A. One, HPB 1688, which had the biggest inhibition zone against L.monocytogenes Scott A, was selected for subsequent analysis. Both ribotyping and DNAsequencing of 16S ribosomal RNA gene demonstrated that the isolate was Lactococcus lactis subsp. lactis . Polymerase chain reaction and nucleotide sequencing revealed that thegenomic DNA of the bean-sprout isolates contained a nisin Z structural gene. In MRS broth,bean-sprout isolate HPB 1688 survived at 3–4·5°C for at least 20 d, grew at 4°Cand produced anti-listerial compoundsat 5°C. When co-cultured with L. monocytogenes in MRS broth, the isolate inhibited thegrowth of L. monocytogenes at 4°C after 14d and at 10°C after 2 d. When co-inoculatedwith 10²cells g⁻¹ of L.monocytogenes on fresh-cut ready-to-eat Caesar salad, L. lactis subsp. lactis (10⁸cells g⁻¹) was able to reduce the number of L. monocytogenes by 1–1·4 logs after storage for 10 d at 7° and 10°C. A bacteriocin-producing Enterococcusfaecium was also able to reduce the numbers of L. monocytogenes onCaesar salad, butdid not act synergistically when co-inoculated with L. lactis subsp. lactis .     

Na/K-ATPase tethers phospholipase C and IP3 receptor into a calcium-regulatory complex

We have shown that the caveolar Na/K-ATPase transmits ouabain signals via multiple signalplexes. To obtain the information on the composition of such complexes, we separated the Na/K-ATPase from the outer medulla of rat kidney into two different fractions by detergent treatment and density gradient centrifugation. Analysis of the light fraction indicated that both PLC-gamma1 and IP3 receptors (isoforms 2 and 3, IP3R2 and IP3R3) were coenriched with the Na/K-ATPase, caveolin-1 and Src. GST pulldown assays revealed that the central loop of the Na/K-ATPase alpha1 subunit interacts with PLC-gamma1, whereas the N-terminus binds IP3R2 and IP3R3, suggesting that the signaling Na/K-ATPase may tether PLC-gamma1 and IP3 receptors together to form a Ca(2+)-regulatory complex. This notion is supported by the following findings. First, both PLC-gamma1 and IP3R2 coimmunoprecipitated with the Na/K-ATPase and ouabain increased this interaction in a dose- and time-dependent manner in LLC-PK1 cells. Depletion of cholesterol abolished the effects of ouabain on this interaction. Second, ouabain induced phosphorylation of PLC-gamma1 at Tyr(783) and activated PLC-gamma1 in a Src-dependent manner, resulting in increased hydrolysis of PIP2. It also stimulated Src-dependent tyrosine phosphorylation of the IP3R2. Finally, ouabain induced Ca(2+) release from the intracellular stores via the activation of IP3 receptors in LLC-PK1 cells. This effect required the ouabain-induced activation of PLC-gamma1. Inhibition of Src or depletion of cholesterol also abolished the effect of ouabain on intracellular Ca(2+).     

Coadministration of interleukin 2 plasmid DNA with combined DNA vaccines significantly enhances the protective efficacy against Mycobacterium tuberculosis

Coadministration of interleukin 2(IL-2) plasmid DNA with combined DNA vaccines enhanced Th1-type cellular responses by producing higher amounts of IFN-gamma with a higher ratio of antigen-specific IgG2a/IgG1. The IFN-gamma specific for Ag85B, MPT64, and MPT83 in this group was 415, 267, and 255 U/ml, respectively, and was 1.6-, 1.8-, and 2.5-fold higher than that of the same vaccine without adding IL-2. The IgG2a/IgG1 ratio for Ag85B, MPT64, and MPT83 was 4, 8, and 4, respectively, upon addition of the genetic adjuvant in the DNA vaccine, which was four times higher for every antigen when IL-2 was not included. Fluorescence activated cell sorter (FACS) analysis showed that, in the presence of IL-2, CD8+ and CD4+ T cells increased significantly, whereas in the absence of the genetic adjuvant, only a mild increase was observed for CD8+ T cells compared to the vector DNA-treated group. Bacterial CFU was reduced to less than 1/100 in the lung and to about 1/10 in the spleen relative to the same combined DNA vaccine without IL-2. The lungs of this group of mice showed much less damage due to an influx of epithelioid macrophages and less lymphocytes. RT-PCR showed that antigen genes could be detected in more organs and for a longer period of time when treated with combined DNA vaccine formulated in IL-2. We suggest that IL-2 enhanced the immunigencity and protective efficacy in immunized mice by improving the Th1-type response and also by prolonging the antigen gene expression in different organs.     

Aberrant IgM signaling promotes survival of transitional T1 B cells and prevents tolerance induction in lupus-prone New Zealand black mice

New Zealand Black (NZB) mice develop a lupus-like syndrome. Although the precise immune defects leading to autoantibody production in these mice have not been characterized, they possess a number of immunologic abnormalities suggesting that B cell tolerance may be defective. In the bone marrow, immature self-reactive B cells that have failed to edit their receptors undergo apoptosis as a consequence of Ig receptor engagement. Splenic transitional T1 B cells are recent bone marrow emigrants that retain these signaling properties, ensuring that B cells recognizing self-Ags expressed only in the periphery are deleted from the naive B cell repertoire. In this study we report that this mechanism of tolerance is defective in NZB mice. We show that NZB T1 B cells are resistant to apoptosis after IgM cross-linking in vitro. Although extensive IgM cross-linking usually leads to deletion of T1 B cells, in NZB T1 B cells we found that it prevents mitochondrial membrane damage, inhibits activation of caspase-3, and promotes cell survival. Increased survival of NZB T1 B cells was associated with aberrant up-regulation of Bcl-2 after Ig receptor engagement. We also show that there is a markedly increased proportion of NZB T1 B cells that express elevated levels of Bcl-2 in vivo and provide evidence that up-regulation of Bcl-2 follows encounter with self-Ag in vivo. Thus, we propose that aberrant cell signaling in NZB T1 B cells leads to the survival of autoreactive B cells, which predisposes NZB mice to the development of autoimmunity.     

Quantile regression models with multivariate failure time data

As an alternative to the mean regression model, the quantile regression model has been studied extensively with independent failure time data. However, due to natural or artificial clustering, it is common to encounter multivariate failure time data in biomedical research where the intracluster correlation needs to be accounted for appropriately. For right-censored correlated survival data, we investigate the quantile regression model and adapt an estimating equation approach for parameter estimation under the working independence assumption, as well as a weighted version for enhancing the efficiency. We show that the parameter estimates are consistent and asymptotically follow normal distributions. The variance estimation using asymptotic approximation involves nonparametric functional density estimation. We employ the bootstrap and perturbation resampling methods for the estimation of the variance-covariance matrix. We examine the proposed method for finite sample sizes through simulation studies, and illustrate it with data from a clinical trial on otitis media.     

中性内肽酶及其抑制剂在心血管疾病过程中的作用

中性内肽酶是一种属于Ⅱ型跨膜蛋白的肽类内切酶,在肽链的氨基端水解疏水氨基酸肽键,灭活心钠素、肾上腺髓质素、血管紧张素、内皮素等心血管活性肽,在高血压病、心力衰竭、动脉粥样硬化和休克等心血管疾病的发病过程中发挥重要作用。