Functional rules for lac repressor�Coperator associations and implications for protein�CDNA interactions

The Lac repressor has been used as a tool to understand protein–DNA recognition for many years. Recent experiments have demonstrated the ability of the Lac repressor to control gene expression in various eukaryotic systems, making the quest for an arsenal of protein–DNA binding partners desirable for potential therapeutic applications. Here, we present the results of the most exhaustive screen of Lac repressor‐DNA binding partners to date, resulting in the elucidation of functional rules for Lac–DNA binding. Even within the confines of a single protein–DNA scaffold, modes of binding of different protein–DNA partners are sufficiently diverse so as to prevent elucidation of generalized rules for recognition for a single protein, much less an entire protein family.     

蚂蚁光顾云南紫胶虫对其天敌紫胶黑虫种群的影响

为了弄清蚂蚁光顾云南紫胶虫Kerria yunnanensis Ouet Hong对其天敌紫胶黑虫Holcocera pulverea Meyr种群的影响,于云南紫胶虫成虫期,对有无蚂蚁光顾的胶被抽样,调查胶被上紫胶黑虫的为害率及种群数量变化。结果显示,紫胶黑虫对有无蚂蚁光顾的胶被的为害率均很高,并逐月增加;其中有蚂蚁光顾的胶被紫胶黑虫的为害率略小于无蚂蚁光顾的胶被。蚂蚁光顾能明显减少紫胶黑虫的种群数量(︱t︱=2.764,df=356,P<0.01),其原因可能是蚂蚁光顾能干扰紫胶黑虫的产卵行为、破坏卵、取食卵和幼虫并且这种保护主要发生在云南紫胶虫幼虫期;云南紫胶虫进入成虫期后,由于紫胶黑虫生活于胶被内,并且产卵于胶被的凹陷处或雄虫胶壳内或雌虫肛突孔处,蚂蚁很少与紫胶黑虫相遇,故蚂蚁光顾对紫胶黑虫每月增长量没有显著影响(︱t︱=0.970,df=161,P>0.05)。紫胶黑虫和蚂蚁相遇的行为反应存在显著差异(χ2=4.781,df=1,P<0.05),蚂蚁对紫胶黑虫有捕食作用。蚂蚁与云南紫胶虫之间存在互利关系。蚂蚁取食云南紫胶虫的蜜露,能降低紫胶黑虫的为害率,并减少紫胶黑虫的种群数量,从而保护云南紫胶虫。     

Mutational analysis of the stability of the H2A and H2B histone monomers

The eukaryotic histone heterodimer H2A-H2B folds through an obligatory dimeric intermediate that forms in a nearly diffusion-limited association reaction in the stopped-flow dead time. It is unclear whether there is partial folding of the isolated monomers before association. To address the possible contributions of structure in the monomers to the rapid association, we characterized H2A and H2B monomers in the absence of their heterodimeric partner. By far-UV circular dichroism, the H2A and H2B monomers are 15% and 31% helical, respectively—significantly less than observed in X-ray crystal structures. Acrylamide quenching of the intrinsic Tyr fluorescence was indicative of tertiary structure. The H2A and H2B monomers exhibit free energies of unfolding of 2.5 and 2.9 kcal mol^− 1, respectively; at 10 μM, the sum of the stability of the monomers is ∼ 60% of the stability of the native dimer. The helical content, stability, and m values indicate that H2B has a more stable, compact structure than H2A. The monomer m values are larger than expected for the extended histone fold motif, suggesting that the monomers adopt an overly collapsed structure. Stopped-flow refolding—initiated from urea-denatured monomers or the partially folded monomers populated at low denaturant concentrations—yielded essentially identical rates, indicating that monomer folding is productive in the rapid association and folding of the heterodimer. A series of Ala and Gly mutations were introduced into H2A and H2B to probe the importance of helix propensity on the structure and stability of the monomers. The mutational studies show that the central α-helix of the histone fold, which makes extensive intermonomer contacts, is structured in H2B but only partially folded in H2A.     

Demethylation of the aryl hydrocarbon receptor repressor as a biomarker for nascent smokers

Epigenetic modifications to peripheral white blood cell DNA occur in response to a wide variety of exposures. In prior work, we and others have shown that broad changes in DNA methylation, particularly at the aryl hydrocarbon receptor repressor (AHRR) locus, occur in samples from subjects with long histories of smoking. However, given the large number of epigenetic changes that occur in response to prolonged smoking, the primacy of the response at AHRR and the sensitivity of these changes to low levels of smoking are not known. Therefore, we examined the association of smoking to genome lymphocyte DNA methylation status in a representative sample of 399 African American youths living in the rural South that includes 72 subjects with less than one half-pack year of exposure. Consistent with our prior findings, we found a stepwise effect of smoking on DNA methylation among youth with relatively brief exposure histories at a CpG residue in AHRR (cg05575921) (FDR corrected p values; 3 × 10⁻⁷ and 0.09 in the male and female samples, respectively) that was identified in previous studies and at which the effects of smoking were significant, even in those subjects with less than one half pack year exposure. We conclude that AHRR demethylation at cg05575921 in peripheral cells may serve as an early, sensitive biomarker for even low levels of exposure to tobacco smoke, providing a non-self-report alternative for nascent exposure to tobacco smoke. We also suggest that the AHRR/AHR pathway may be functional in the response of peripheral white blood cells to tobacco smoke exposure.     

紫胶虫的生物学研究

紫胶虫Laccifer lacca(Kerr)Targ.在云南自然分布区一年发生两代,各世代有涌散、固定、泌胶、泌蜡和排泄蜜露等活动。由于幼虫在饥饿状态下的存活期较短,迁移能力较差,必须及时实行人工放养和科学管理,才能获得紫胶高产。本文报道了紫胶虫各世代的泌胶量、生殖力和性比,并提出发展紫胶生产的建议。     

Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

Cellular repressor of E1A-stimulated genes regulates vascular endothelial cell migration by the ILK/AKT/mTOR/VEGF(165) signaling pathway

The migration of vascular endothelial cells plays a critical role in a variety of vascular physiological and pathological processes, such as embryonic development, angiogenesis, wound healing, re-endothelialization, and vascular remodeling. This study clarified the role and mechanism of a new vascular homeostasis regulator, Cellular repressor of E1A-stimulated genes (CREG), in the migration of primary human umbilical vein endothelial cells (HUVECs). A wound healing assay and transwell migration model showed that upregulation of CREG expression induced HUVEC migration and it was positively correlated with the expression of vascular endothelial growth factor. Furthermore, wild type integrin-linked kinase reversed the poor mobility of CREG knock-down HUVECs; in contrast, kinase-dead integrin-linked kinase weakened the migration of HUVECs. We also studied the effect of CREG on HUVEC migration by the addition of an mTOR inhibitor, recombinant vascular endothelial growth factor₁₆₅, neutralizing antibody of vascular endothelial growth factor₁₆₅ and AKT siRNA, and we concluded that CREG induces endothelial cell migration by activating the integrin-linked kinase/AKT/mTOR/VEGF₁₆₅ signaling pathway.     

The MADS box gene, FOREVER YOUNG FLOWER, acts as a repressor controlling floral organ senescence and abscission in Arabidopsis

The ectopic expression of a MADS box gene FOREVER YOUNG FLOWER (FYF) caused a significant delay of senescence and a deficiency of abscission in flowers of transgenic Arabidopsis. The defect in floral abscission was found to be due to a deficiency in the timing of cell separation of the abscission zone cells. Down-regulation of INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) may contribute to the delay of the floral abscission in 35S:FYF flowers. FYF was found to be highly expressed in young flowers prior to pollination and was significantly decreased after pollination, a pattern that correlated with its function. Ethylene insensitivity in senescence/abscission and the down-regulation of ETHYLENE RESPONSE DNA-BINDING FACTOR 1 (EDF1) and EDF2, downstream genes in the ethylene response, in 35S:FYF Arabidopsis suggested a role for FYF in regulating senescence/abscission by suppressing the ethylene response. This role was further supported by the fact that 35S:FYF enhanced the delay of flower senescence/abscission in ethylene response 1 (etr1), ethylene-insensitive 2 (ein2) and constitutive triple response 1 (ctr1) mutants, which have defects in upstream genes of the ethylene signaling pathway. The presence of a repressor domain in the C-terminus of FYF and the enhancement of the delay of senescence/abscission in FYF+SRDX (containing a suppression motif) transgenic plants suggested that FYF acts as a repressor. Indeed, in FYF-DR+VP16 transgenic dominant-negative mutant plants, in which FYF was converted to a potent activator by fusion to a VP16-AD motif, the senescence/abscission of the flower organs was significantly promoted, and the expression of BOP2, IDA and EDF1/2 was up-regulated. Our data suggest a role for FYF in controlling floral senescence/abscission by repressing ethylene responses and regulating the expression of BOP2 and IDA in Arabidopsis.     

Apoptosis repressor with caspase recruitment domain (ARC) inhibits myogenic differentiation

Apoptosis repressor with caspase recruitment domain (ARC), an anti-apoptotic protein, is highly expressed in differentiated heart and skeletal muscle. Apoptosis and differentiation share numerous common pathways; therefore, we examined the impact of ARC on H9c2-myoblast differentiation. We demonstrate that ARC expression levels increase and stabilize upon differentiation. ARC-overexpression in pre-differentiated H9c2-cells suppresses differentiation; indicated by increased myotube formation, nuclear fusion and expression of the differentiation markers myogenin and troponin-T. ARC-overexpression inhibited myoblast differentiation associated caspase-3 activation, suggesting ARC inhibits myogenic differentiation through caspase inhibition. In summary, we show a novel role for ARC in the regulation of muscle differentiation.