Conformational Analysis of a Hybrid DNA-RNA Double Helical Oligonucleotide in Aqueous Solution: d(CG)r(CG)d(CG) Studied by 1D- and 2D-1H NMR Spectroscopy

Abstract

The double helical structure of the self-complementary DNA-RNA-DNA hybrid d(CG)r(CG) d(CG) was studied in solution by 500 MHz ¹H-NMR spectroscopy. The non-exchangeable base protons and the (deoxy)ribose H1′, H2′ and H2″ protons were unambiguously assigned using 2D-J-correlated (COSY) and 2D-NOE (NOESY) spectroscopy techniques. A general strategy for the sequential assignment of ¹H-NMR spectra of (double) helical DNA and RNA fragments by means of 2D-NMR methods is presented.

Conformational analysis of the sugar rings of d(CG)r(CG)d(CG) at 300 K shows that the central ribonucleotide part of the helix adopts an A-type double helical conformation. The 5′- and 3′-terminal deoxyribose base pairs, however, take up the normal DNA-type conformation. The A-to-B transition in this molecule involves only one (deoxyribose) base pair. It is shown that this A-to-B conformational transition can only be accomodated by two specific sugar pucker combinations for the junction base pair, i.e. N·S (C3′-endo-C2′-endo, 60%, where the pucker given first is that assigned to the junction nucleotide residue of the strand running 5′ → 3′ from A-RNA to B-DNA) and S·S (C2′-endo-C2′-endo, 40%).     

Inhibition of p38/CREB phosphorylation and COX-2 expression by olive oil polyphenols underlies their anti-proliferative effects

We investigated the anti-proliferative effects of an olive oil polyphenolic extract on human colon adenocarcinoma cells. Analysis indicated that the extract contained hydroxytyrosol, tyrosol and the various secoiridoid derivatives, including oleuropein. This extract exerted a strong inhibitory effect on cancer cell proliferation, which was linked to the induction of a G2/M phase cell cycle block. Following treatment with the extract (50 microg/ml) the number of cells in the G2/M phase increased to 51.82+/-2.69% relative to control cells (15.1+/-2.5%). This G2/M block was mediated by the ability of olive oil polyphenols (50 microg/ml) to exert rapid inhibition of p38 (38.7+/-4.7%) and CREB (28.6+/-5.5%) phosphorylation which led to a downstream reduction in COX-2 expression (56.9+/-9.3%). Our data suggest that olive oil polyphenols may exert chemopreventative effects in the large intestine by interacting with signalling pathways responsible for colorectal cancer development.     

Synaptotagmin I and IX function redundantly in controlling fusion pore of large dense core vesicles

Synaptotagmins (Syts) constitute a large family of at least 16 members and individual Syt isoforms exhibit distinct Ca²⁺-binding properties and subcellular localization. It remains to be demonstrated whether multiple Syt isoforms can function independently or cooperatively on certain type of vesicle. In the current study, we have developed NPY-pHluorin to specifically assess exocytosis of large dense core vesicles (LDCVs) and studied the requirement of Syt I and Syt IX for LDCV exocytosis in PC12 cells. We found that down-regulation of both Syt I and Syt IX resulted in a significant loss of Ca²⁺-dependent LDCV exocytosis. Moreover, our results suggest Syt I and Syt IX play redundant role in controlling the choice of fusion modes. Down-regulation of both Syt I and Syt IX renders more fusion in the kiss-and-run mode. We conclude that Syt I and Syt IX function redundantly in Ca²⁺-sensing and fusion pore dilation on LDCVs in PC12 cells.     

Mutation,selection, and ancestry in branching models: a variational approach

We consider the evolution of populations under the joint action of mutation and differential reproduction, or selection. The population is modelled as a finite-type Markov branching process in continuous time, and the associated genealogical tree is viewed both in the forward and the backward direction of time. The stationary type distribution of the reversed process, the so-called ancestral distribution, turns out as a key for the study of mutation–selection balance. This balance can be expressed in the form of a variational principle that quantifies the respective roles of reproduction and mutation for any possible type distribution. It shows that the mean growth rate of the population results from a competition for a maximal long-term growth rate, as given by the difference between the current mean reproduction rate, and an asymptotic decay rate related to the mutation process; this tradeoff is won by the ancestral distribution. We then focus on the case when the type is determined by a sequence of letters (like nucleotides or matches/mismatches relative to a reference sequence), and we ask how much of the above competition can still be seen by observing only the letter composition (as given by the frequencies of the various letters within the sequence). If mutation and reproduction rates can be approximated in a smooth way, the fitness of letter compositions resulting from the interplay of reproduction and mutation is determined in the limit as the number of sequence sites tends to infinity. Our main application is the quasispecies model of sequence evolution with mutation coupled to reproduction but independent across sites, and a fitness function that is invariant under permutation of sites. In this model, the fitness of letter compositions is worked out explicitly. In certain cases, their competition leads to a phase transition.      

Complete mitochondrial genome of the stonefly Cryptoperla stilifera Sivec (Plecoptera: Peltoperlidae) and the phylogeny of Polyneopteran insects

We present the complete mitogenome of a stonefly, Cryptoperla stilifera Sivec (Plecoptera; Peltoperlidae). The mitogenome was a circular molecule consisting of 15,633 nucleotides, 37 genes and a A + T-rich region. C. stilifera mitogenome was similar to Pteronarcys princeps mitogenome (Plecoptera; Pteronarcyidae). All transfer RNA genes (tRNAs) had typical cloverleaf secondary structures except for trnSer (AGN), where the stem-loop structure of the dihydrouridine (DHU) arm was missing. The A + T-rich region of C. stilifera had two stem-loops and each had two interlink. Three conserved sequence blocks (CSBs) were present in the A + T-rich regions of C. stilifera, Peltoperla tarteri and Peltoperla arcuata. Moreover, many polynucleotide stretches (Poly N, N = A, T and C) in the A + T-rich region of C. stilifera Phylogenetic relationships of Polyneopteran species were constructed based on the nucleotide sequences of 13 protein coding genes (PCGs). Both maximum likelihood (ML) and Bayesian inference (BI) analyses supported Grylloblattodea as the sister group to Plecoptera + Dermaptera and Embiidina and Phasmatodea as sister groups.     

Behavioural differences in sub-adult female mice exposed to a murine elevated plus-maze: correlated effects of selection for high litter size

The present experiment compared the anxiety- and activity-related behaviour of sub-adult females from a mouse strain selected for over 106 generations for high litter size with that of a randomly selected control strain, to illuminate possible differences in their ability to cope with exposure to a novel environment. Selected for large litter size, the H-strain has an average litter size of 21 pups, whereas the randomly bred C-strain has an average litter size of 10 pups. The elevated plus-maze was used to measure the behaviour of the mice expressed in response to novelty. The results are described and discussed in relation to the ability to cope with novel environmental challenges. In the elevated plus-maze, the H-strain was significantly more anxious (having a lower percentage of entries into and percentage of time spent on open arms) and less active (having a lower number of entries into closed arms, and a lower total number of arm entries) than the C-strain. Thus, there were clear anxiety- and activity-related differences between the strains, which may be related to selection for large litter size. A tentative hypothesis is presented whereby selection for large litter size may accelerate adaptation to the home environment, but decrease the ability of selected animals to cope with exposure to novelty.     

不同发育阶段大鼠不同大动脉光镜及激光共聚焦扫描显微镜观察

应用光镜和激光共聚焦扫描显微镜对初生、3月龄、24月龄大鼠升主动脉、胸主动脉、腹主动脉和髂总动脉的管壁面积、管腔面积、中膜厚度和弹性膜进行了观察。结果分析显示,大鼠升主动脉、胸主动脉、腹主动脉和髂总动脉显微结构随发育、衰老发生明显的变化,单位面积内弹性膜荧光强度、管壁面积、管腔面积和中膜厚度均呈增大趋势,这种结构变化是导致动脉功能产生相应变化的主要因素。     

双歧杆菌的完整肽聚糖对实验性大肠癌NF-κB和IκB α的影响

目的 :探索分叉双歧杆菌的完整肽聚糖 (WPG)的体内抑瘤途径。方法 :以激光共聚焦显微镜和免疫组化检测了大肠癌裸鼠移植瘤IκBα的含量以及NF κB的活化状况。结果 :大肠癌裸鼠移植瘤经WPG处理后 ,其IκBα的平均荧光强度明显高于肿瘤对照组 (P <0 0 1) ;而NF κB的活化状态则相反 ,WPG注射组大肠癌NF κB的阳性细胞密度显著低于肿瘤对照组 (P <0 0 1)。结论 :分叉双歧杆菌的WPG体内能明显抑制大肠癌IκBα的降解 ,最终阻抑NF κB的活化。