Nucleosome-like Complex of the Histone from the Hyperthermophile Methanopyrus Kandleri (MkaH) with Linear DNA

Abstract

The MkaH protein from the archaeon Methanopyrus kandleri, an unusual assembly of two histone-fold domains in a single polypeptide chain, demonstrates high structural similarity to eukaryal histones. We studied the DNA binding and self-association properties of MkaH by means of the electrophoretic mobility shift assay (EMSA), electron microscopy (EM), chemical cross-linking, and analytical gel filtration. EMSA showed an increased mobility of linear DNA complexed with MkaH protein with a maximum at a protein-DNA weight ratio (Rw) of ≈3; the mobility decreased at higher protein concentration. EM of the complexes formed at Rw ≤ 3 revealed formation of isometric loops encompassing 71 +/- 7 bp of DNA duplex. At high values of Rw (≥9) thickened compact nucleoprotein structures were observed; no individual loops were seen within the complexes. Gel filtration chromatography and chemical fixation indicated that in the absence of DNA the dominant form of the MkaH in solution, unlike other archaeal histones, is a stable dimer (pseudo-tetramer of the histone-fold domain) apparently resembling the eukaryal (H3-H4)₂ tetramer. Similarly, dimers are the dominant form of the protein interacting with DNA. The properties of MkaH supporting the assignment of its intermediate position between other archaeal and eukaryal histones are discussed.     

Sequence-directed Mapping of Nucleosome Positions

Abstract

A nucleosome DNA sequence probe is designed that combines recently derived RR/YY counter-phase and AA/TT in-phase periodical patterns. A simple nucleosome mapping procedure is introduced for prediction of the nucleosome positions in the sequence of interest, to serve as a guide for experimental studies of the chromatin structure.     

Systematic Characterization of Myocardial Inflammation,Repair, and Remodeling in a Mouse Model of Reperfused Myocardial Infarction

Mouse models of myocardial infarction are essential tools for the study of cardiac injury, repair, and remodeling. Our current investigation establishes a systematic approach for quantitative evaluation of the inflammatory and reparative response, cardiac function, and geometry in a mouse model of reperfused myocardial infarction. Reperfused mouse infarcts exhibited marked induction of inflammatory cytokines that peaked after 6 hr of reperfusion. In the infarcted heart, scar contraction and chamber dilation continued for at least 28 days after reperfusion; infarct maturation was associated with marked thinning of the scar, accompanied by volume loss and rapid clearance of cellular elements. Echocardiographic measurements of end-diastolic dimensions correlated well with morphometric assessment of dilative remodeling in perfusion-fixed hearts. Hemodynamic monitoring was used to quantitatively assess systolic and diastolic function; the severity of diastolic dysfunction following myocardial infarction correlated with cardiomyocyte hypertrophy and infarct collagen content. Expression of molecular mediators of inflammation and cellular infiltration needs to be investigated during the first 72 hr, whereas assessment of dilative remodeling requires measurement of geometric parameters for at least four weeks after the acute event. Rapid initiation and resolution of the inflammatory response, accelerated scar maturation, and extensive infarct volume loss are important characteristics of infarct healing in mice.     

Distinct roles for ROCK1 and ROCK2 in the regulation of cell detachment

This study, using mouse embryonic fibroblast (MEF) cells derived from ROCK1^−/− and ROCK2^−/− mice, is designed to dissect roles for ROCK1 and ROCK2 in regulating actin cytoskeleton reorganization induced by doxorubicin, a chemotherapeutic drug. ROCK1^−/− MEFs exhibited improved actin cytoskeleton stability characterized by attenuated periphery actomyosin ring formation and preserved central stress fibers, associated with decreased myosin light chain 2 (MLC2) phosphorylation but preserved cofilin phosphorylation. These effects resulted in a significant reduction in cell shrinkage, detachment, and predetachment apoptosis. In contrast, ROCK2^−/− MEFs showed increased periphery membrane folding and impaired cell adhesion, associated with reduced phosphorylation of both MLC2 and cofilin. Treatment with inhibitor of myosin (blebbistatin), inhibitor of actin polymerization (cytochalasin D), and ROCK pan-inhibitor (Y27632) confirmed the contributions of actomyosin contraction and stress fiber instability to stress-induced actin cytoskeleton reorganization. These results support a novel concept that ROCK1 is involved in destabilizing actin cytoskeleton through regulating MLC2 phosphorylation and peripheral actomyosin contraction, whereas ROCK2 is required for stabilizing actin cytoskeleton through regulating cofilin phosphorylation. Consequently, ROCK1 and ROCK2 can be functional different in regulating stress-induced stress fiber disassembly and cell detachment.     

咪喹莫特对慢性哮喘模型小鼠肺组织中基质金属蛋白酶-9-表达的影响

目的：观察Toll样受体7配体咪喹莫特对慢性哮喘小鼠模型气道重塑及肺组织中基质金属蛋白酶MMP-9表达的影响。方法：36只BALB／c小鼠按随机原则分成正常对照组、哮喘模型组、咪喹莫特组,每组12只。通过卵蛋白致敏,气道激发8周,末次激发24h后,检测各组小鼠气道反应性,HE染色观察气道炎症变化;Masson三色染色观察气道纤维化的改变;real-timePCR和western—blot分别检测肺组织中MMP-9的mRNA和蛋白表达。结果：慢性哮喘组小鼠气道炎症、气道高反应性和气道重塑较正常对照小鼠明显加重,而咪喹莫特组小鼠模型的气道炎症和气道反应性及气道重塑均较哮喘模型组小鼠减少或降低。慢性哮喘组小鼠肺组织MMP-9的mRNA和蛋白水平均较正常对照小鼠明显增加（P〈0．05）,而咪喹莫特治疗可显著降低哮喘小鼠肺组织MMP-9的mRNA和蛋白水平（P〈0．05）。结论：咪喹莫特能够显著抑制慢性哮喘小鼠模型的气道炎症、降低气道高反应性并减轻气道重塑,这可能与其抑制MMP-9的表达有关。     

古菌ESCRT系统研究进展

内体分拣转运复合体（ESCRT,endosomal sorting complex required for transport）曾被认为是真核生物特有的系统,涉及膜重塑、泛素化蛋白质分拣等重要细胞生命过程。近年的研究显示,TACK（包括Thaumarchaeota、Aigarchaeota、Crenarchaeota和Korarchaeota门）古菌超门中存在着一类与分泌膜囊泡、古菌病毒出胞以及细胞分裂过程等膜重塑过程相关的细胞分裂（Cdv,cell division）系统,该系统中的CdvB和CdvC是真核生物ESCRT-III和Vps4的同源蛋白,提示真核生物ESCRT系统可能起源自古菌。然而,由于TACK古菌中缺少真核生物ESCRT系统的其他关键成分,这一假设仍有争议。最近发现的阿斯加德（Asgard）古菌是一类被认为与真核生物最近缘的古菌,其基因组具有较完整的ESCRT相关蛋白的编码基因,提示真核生物的ESCRT很可能起源于阿斯加德古菌。本文首先简要介绍真核生物ESCRT系统的组成及生物学功能,然后分别总结TACK古菌的Cdv系统和阿斯加德古菌的ESCRT系统的研究进展,重点讨论它们的组成及生物学功能,为进一步了解古菌ESCRT系统与真核生物起源的关系提供参考。