可视化动缘探测系统检测心肌细胞舒缩功能

目的:分离成年大鼠心室肌细胞,利用可视化动缘探测系统检测其收缩/舒张功能.方法:常规酶解法分离制备钙耐受心肌细胞;用Ca2 荧光探剂fura-2/AM(0.5μmol/L)负载心肌细胞30 min(25℃)后,以含氧台氏液灌流并予电场刺激(0.5 Hz,5 ms),采用动缘探测系统检测心肌细胞收缩/舒张功能及细胞内荧光信号变化.结果:可视化动缘探测系统可实时同步观察和记录心肌细胞机械功能和细胞内钙瞬变变化,并可计算得到细胞收缩/舒张和钙瞬变的变化幅度、时程及速率等一系列指标,从而直接反映细胞水平的心肌功能改变.结论:可视化动缘探测系统为人们提供了一个在单心肌细胞水平探讨心肌肌源性功能及其改变的新方法,是当今心血管研究的一项重要技术.     

次声暴露对大鼠脾、肝脏某些酶活性的影响

目的:观察8 Hz,130 dB次声暴露不同时间对大鼠脾、肝脏某些酶活性的影响.方法:35只SD大鼠随机分为5组,即对照组,1周,2周,3周,4周组.每天次声暴露1次,每次2 h.实验后,观察大鼠脾、肝脏组织中MAO,GSH-px,SOD活性和MDA含量的变化.结果:大鼠脾脏MAO活性1周,2周时显著增高(P＜0.01),3周下降,4周时又显著增加(P＜0.05).肝脏组织MAO活性变化不明显(P＞0.05).脾脏组织中GSH-px活性在4周时明显增高(P＜0.05),肝脏组织中GSH-px活性在1周时就有显著性增高(P＜0.05).脾脏SOD活性在1周至4周均有显著性增高(P＜0.05).肝脏组织在实验期变化不明显(P＞0.05).脾脏组织中MDA含量在3周至4周时有显著性增高(P＜0.05).肝脏组织在1至2周时有非常显著的增高(P＜0.01),在3周时下降,到4周时又显著高于对照组(P＜0.05).结论:8Hz,130 dB次声暴露,大鼠脾、肝脏组织活性氧自由基、脂质过氧化物增高,抗氧化能力降低,造成对组织的损伤.     

The cell cycle related apoptotic susceptibility to arsenic trioxide is associated with the level of reactive oxygen species

Double staining flow cytometry was performed using 7-amino actinomycin D and 6-carboxy-2‘,7‘-dichlorodihydrofluorescein diacetate, to detect the level fluctuation of reactive oxygen species (ROS) during the cell cycle of normal NB4 cells. Our results showed that NB4 cells possessed higher level of ROS in G2/M phase than in G1 and S phases. Double staining flow cytometry, with TdT mediated dUTP nick end labeling (Tunel) and propidium iodide(PI), indicated that As2O3 (2μM) could induce apoptosis in NB4 cells prevailingly from G2/M phase, and this efficacy was enhanced upon co-administration of 2, 3-dimethoxy-1, 4-naphthoquinone (DMNQ) (2.5μM) which could produce the endogenous ROS. These results suggested that different ROS level in different cell cycle phases of NB4 cells might determin the selective induction of G2/M apoptosis and the cells‘ susceptibility to apoptosis by As2O3.     

Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset

A method of supervised classification using two available structure templates was applied to investigate the possible heterogeneity existing in a large cryo-EM dataset of an Escherichia coli 70S ribosome-EF-G complex. Two subpopulations showing the ribosome in distinct conformational states, related by a ratchet-like rotation of the 30S subunit with respect to the 50S subunit, were extracted from the original dataset. The possible presence of additional intermediate states is discussed.     

Pxl1p, a paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and Rho1p functions during polarized growth

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the "ancient paxillin" in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.     

RNA interference inhibition of Mus81 reduces mitotic recombination in human cells

Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.     

3'-modified oligonucleotides by reverse DNA synthesis

Reverse DNA oligonucleotide synthesis (i.e. from 5′→3′) is a strategy that has yet to be exploited fully. While utilized previously for the construction of alternating 3′-3′- and 5′-5′-linked antisense oligonucleotides, the use of nucleoside 5′-phosphoramidites has not generally been used for the elaboration of (modified) oligonucleotides. Presently, the potential of reverse oligonucleotide synthesis for the facile synthesis of 3′-modified DNAs is illustrated using a phosphoramidite derived from tyrosine. The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligonucleotide resulting from the proteinase K digestion of the vaccinia topoisomerase I–DNA covalent complex. The results confirm the nature of the structure previously assigned to this product, and establish the facility with which proteinase K is able to complete the digestion of the polypeptide backbone of the DNA oligonucleotide-linked topoisomerase I.     

Chemical identification of<Emphasis Type="Italic"> N</Emphasis>-acyl homoserine lactone quorum-sensing signals produced by<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis> strains in defined medium

The N-acyl homoserine lactone (AHL) quorum-sensing signals produced by Sinorhizobium meliloti strains AK631 and 1021 when cultured in a defined glucose-nitrate medium were identified by gas chromatography/mass spectrometry (GC/MS) and electrospray ionization tandem mass spectrometry (ESI MS/MS). Both strains synthesized several long-chain AHLs. Defined medium cultures of strain AK631 synthesized a complex mixture of AHLs with short acyl side chains. Strain 1021 produced no short-chain AHLs when grown on defined medium and made a somewhat different set of long-chain AHLs than previously reported for cultures in rich medium. While the two strains produced several AHLs in common, the differences in AHLs produced suggest that there may be significant differences in their patterns of quorum-sensing regulation.     

Identification and characterization of a novel cross-link lesion in d(CpC) upon 365-nm irradiation in the presence of 2-methyl-1,4-naphthoquinone

We report the isolation and characterization for the first time of a cross-link lesion between two adjacent cytosines from the 2-methyl-1,4-naphthoquinone (menadione)-sensitized 365-nm irradiation of d(CpC). Electrospray ionization mass spectrometry (ESI-MS), tandem MS and ¹H NMR results indicate that the cross-link occurs between the C5 carbon atom of one cytosine and the N⁴ nitrogen atom of the other cytosine. Furthermore, we synthesized d(CpC) with a ¹⁵N being incorporated on the amino group of either of the two cytosines. We then irradiated the two ¹⁵N-labeled dinucleoside monophosphates, isolated the cross-link products and characterized them by MS and multi-stage tandem MS. The latter results established unambiguously that the N⁴ nitrogen atom of the 3′-nucleobase is involved in the covalent bond formation between the two cytosines. This, in combination with two-dimensional nuclear Overhauser effect spectroscopy (NOESY) results, demonstrates that the cross-link arises from the formation of a covalent bond between the C5 carbon atom of the 5′ cytosine and the N⁴ nitrogen atom of the 3′ cytosine. We also show that the solution pH has a significant effect on the formation of the cross-link lesion, which supports that the deprotonation at the exocyclic amino group of cytosine cation radical is essential for the formation of the cross-link lesion.     

镉负荷大鼠一氧化氮和肿瘤坏死因子-α释放在介导多器官机能活动障碍中的作用

选择健康SD雄性成年大鼠36只,随机分成对照组(C组)、镉负荷中剂量组(M组)、镉负荷高剂量组(H组).将分析纯CdCl2·2.5H2O用生理盐水稀释成含镉0.4 mg/ml浓度的注射溶液,高压灭菌.M组和H组大鼠每天分别按含镉0.5和1.0 mg/kg体重腹腔注射染毒,C组用同样方法注射与H组同等剂量的生理盐水,进行急性镉负荷实验,连续观察7 d.研究急性镉负荷对大鼠血液及几种组织中一氧化氮(NO)自由基、肿瘤坏死因子-α(TNF-a)变化的影响及作用.结果显示在整个实验期内,镉负荷大鼠体重与对照组比较明显下降;睾丸、心脏和肝脏组织中的镉含量极显著上升,并随镉负荷剂量和时间而增加;血浆NO水平M组虽高于对照组,但差异不显著,而H组极显著高于对照组,M和H组血浆TNF-α明显高于对照组;在整个实验期内,镉负荷大鼠睾丸、心脏和肝脏组织匀浆中NO较对照组高或明显高于对照组,睾丸和心脏组织匀浆中TNF-a也均高于或明显高于对照组,但肝脏中的TNF-a三组间没有差异.结果提示,镉负荷诱发NO、TNF-α大量释放在导致大鼠多种器官机能活动障碍发生过程中可能起重要作用.