Biochemical and genetic characterization of three hamster cell mutants resistant to diphtheria toxin

We describe here three different hamster cell mutants which are resistant to diphtheria toxin and which provide models for investigating some of the functions required by the toxin inactivates elongation factor 2 (EF-2). Cell-free extracts from mutants Dtx(r)-3 was codominant. The evidence suggests that the codominant phenotype is the result of a mutation in a gene coding for EF-2. The recessive phenotype might arise by alteration of an enzyme which modifies the structure of EF-2 so that it becomes a substrate for reaction with the toxin. Another mutant, Dtx(r)-2, contained EF-2 that was sensitive to the toxin and this phenotype was recessive. Pseudomonas aeruginosa exotoxin is known to inactivate EF-2 as does diphtheria toxin and we tested the mutants for cross-resistance to pseudomonas exotoxin. Dtx(r)-1 and Dtx(r)-3 were cross-resistant while Dtx(r)-2 was not. It is known that diphtheria toxin does not penetrate to the cytoplasm of mouse cells and that these cell have a naturally occurring phenotype of diphtheria toxin resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance. We fused each of the mutants with mouse 3T3 cells and measured the resistance of the hybrid cells to diphtheria toxin. Intraspecies hybrids containing the genome of mutants Dtx(r)-1 and Dtx(r)-3 had some resistance while those formed with Dtx(r)-2 were as sensitive as hybrids derived from fusions between wild-type hamster cells and mouse 3T3 cells.     

The effect of the elongation of the proximal aorta on the estimation of the aortic wall distensibility

The compliance of the proximal aortic wall is a major determinant of cardiac afterload. Aortic compliance is often estimated based on cross-sectional area changes over the pulse pressure, under the assumption of a negligible longitudinal stretch during the pulse. However, the proximal aorta is subjected to significant axial stretch during cardiac contraction. In the present study, we sought to evaluate the importance of axial stretch on compliance estimation by undertaking both an in silico and an in vivo approach. In the computational analysis, we developed a 3-D finite element model of the proximal aorta and investigated the discrepancy between the actual wall compliance to the value estimated after neglecting the longitudinal stretch of the aorta. A parameter sensitivity analysis was further conducted to show how increased material stiffness and increased aortic root motion might amplify the estimation errors (discrepancies between actual and estimated distensibility ranging from − 20 to − 62%). Axial and circumferential aortic deformation during ventricular contraction was also evaluated in vivo based on MR images of the aorta of 3 healthy young volunteers. The in vivo results were in good qualitative agreement with the computational analysis (underestimation errors ranging from − 26 to − 44%, with increased errors reflecting higher aortic root displacement). Both the in silico and in vivo findings suggest that neglecting the longitudinal strain during contraction might lead to severe underestimation of local aortic compliance, particularly in the case of women who tend to have higher aortic root motion or in subjects with stiff aortas.

     

SHP2 Drives Adaptive Resistance to ERK Signaling Inhibition in Molecularly Defined Subsets of ERK-Dependent Tumors

1. Download high-res image (238KB)
2. Download full-size image

     

Histone modifications as a pathogenic mechanism of colorectal tumorigenesis

Epigenetic regulation of gene expression has provided colorectal cancer (CRC) pathogenesis with an additional trait during the past decade. In particular, histone post-translational modifications set up a major component of this process dictating chromatin status and recruiting non-histone proteins in complexes formed to "handle DNA". In CRC, histone marks of aberrant acetylation and methylation levels on specific residues have been revealed, along with a plethora of deregulated enzymes that catalyze these reactions. Mutations, deletions or altered expression patterns transform the function of several histone-modifying proteins, further supporting the crucial role of epigenetic effectors in CRC oncogenesis, being closely associated to inactivation of tumor suppressor genes. Elucidation of the biochemical basis of these new tumorigenic mechanisms allows novel potential prognostic factors to come into play. Moreover, the detection of these changes even in early stages of the multistep CRC process, along with the reversible nature of these mechanisms and the technical capability to detect such alterations in cancer cells, places this group of covalent modifications as a further potential asset for clinical diagnosis or treatment of CRC. This review underlines the biochemistry of histone modifications and the potential regulatory role of histone-modifying proteins in CRC pathogenesis, to date. Furthermore, the underlying mechanisms of the emerging epigenetic interplay along with the chemical compounds that are candidates for clinical use are discussed, offering new insights for further investigation of key histone enzymes and new therapeutic targets.     

ANTIPYRETIC ACTION OF DEXAMETHASONE ON EGTAZIC ACIDINDUCED FEVER IN RABBITS

本文用脑室灌注和Ｆｕｒａ２测定细胞内游离钙技术观察了地塞米松（ｄｅｘａｍｅｔｈａｓｏｎｅ,ＤＥＸ）对家兔乙二醇双（２氨基乙醚）四乙酸性发热效应和下丘脑细胞内游离钙浓度（［Ｃａ２＋］ｉ）的影响,借此深入探讨地塞米松解热作用的中枢机制。结果发现：脑室灌注乙二醇双（２氨基乙醚）四乙酸（０６ｎｍｏｌ）引起家兔结肠温度明显升高,静脉注射地塞米松（５ｍｇ／ｋｇ）显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,地塞米松（６０～１２０μｍｏｌ／Ｌ）并不影响下丘脑细胞内［Ｃａ２＋］ｉ,而事先脑室灌注抑制基因转录的放线菌素Ｄ（３ｎｍｏｌ）则完全取消了地塞米松对乙二醇双（２氨基乙醚）四乙酸性发热的解热作用。这些结果提示：地塞米松显著抑制家兔乙二醇双（２氨基乙醚）四乙酸性发热,其机制与地塞米松激活脑内某些基因的表达有关,而与下丘脑神经细胞跨膜钙离子流无关。     

Instant success for Instant Pharmacology

Instant Pharmacology (1999). Saeb-Parsy K, Assomull RG, Kahn FZ, Saeb-Parsy K, and Kelly E. John Wiley and Sons 349 pp. £19.99 pbk; ISBN 0471976393 © 1999 John Wiley & Sons, Inc.     

人眼微光下夜近视机制的研究

本文利用激光散斑屈光测试系统详细探讨了夜近视(night myopia)的产生机制。夜近视在环境亮度降低到产生暗视觉时出现,并随亮度继续降低而增大,平均值为1.35D。实验证明眼睛球差和色差不是造成夜近视的主要因素。夜近视数值在暗适应过程中呈增大的趋势。在暗视觉时,由于视野中缺少适宜的刺激,眼睛处于暗焦(dark focus)休止状态,这是造成夜近视的主要原因。     

Immune regulation of bone loss by Th17 cells

A significant macrophage and T-cell infiltrate commonly occurs in inflammatory joint conditions such as rheumatoid arthritis that have significant bone destruction. Cytokines produced by activated macrophages and T cells are implicated in arthritis pathogenesis and are involved in osteoclast-mediated bone resorption. The scope of the present review is to analyze current knowledge and to provide a better understanding of how macrophage-derived factors promote the differentiation of a novel T-helper subset (Th17) that promotes osteoclast formation and activation.     

Leukotriene B4 activates intracellular calcium and augments human osteoclastogenesis

Introduction

Bone erosion in inflammatory arthritis depends on the recruitment and activation of bone resorbing cells, the osteoclasts. Interleukin-23 (IL-23) has been primarily implicated in mediating inflammatory bone loss via the differentiation of Th17 receptor activator of nuclear factor κB ligand (RANKL)–producing cells. In this article, we describe a new role of IL-23 in activating the synthesis and production of leukotriene B4 (LTB4) in innate immune cells.

Methods

We utilized whole blood–derived human peripheral blood mononuclear cells (PBMCs), differentiated them towards an osteoclast lineage and then performed immunofluorescence and cytochemical staining to detect the expression of LTB4-associated receptors and enzymes such as phospholipase A2, 5-lipoxygenase and leukotriene A4 hydrolase, as well as the presence of tartrate-resistant acid phosphatase (TRAP) and F-actin rings on fully mature osteoclasts. We used enzyme immunoassays to measure LTB4 levels in culture media derived from IL-23-treated human PBMCs. We used real-time calcium imaging to study the effect of leukotrienes and requirements of different calcium sources and signaling proteins in activating intracellular calcium flux using pharmacological inhibitors to phospholipase C ({"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122), membrane calcium channels (2-APB) and phosphatidylinositol 3-kinase (Wortmannin) and utilized qPCR for gene expression analysis in macrophages and osteoclasts.

Results

Our data show that LTB4 engagement of BLT1 and BLT2 receptors on osteoclast precursors leads to activation of phospholipase C and calcium release–activated channel–mediated intracellular calcium flux, which can activate further LTB4 autocrine production. IL-23-induced synthesis and secretion of LTB4 resulted in the upregulation of osteoclast-related genes NFATC1, MMP9, ACP5, CTSK and ITGB3 and the formation of giant, multinucleated TRAP⁺ cells capable of F-actin ring formation. These effects were dependent on Ca²⁺ signaling and were completely inhibited by BLT1/BLT2 and/or PLC and CRAC inhibitors.

Conclusions

In conclusion, IL-23 can initiate osteoclast differentiation independently from the RANK-RANKL pathway by utilizing Ca²⁺ signaling and the LTB4 signaling cascade.     

瓜类枯萎病研究进展(综述)

瓜类枯萎病是由尖孢镰刀菌Fusarium oxysporum侵染引起,在全球大部分瓜类产区均有发生。该病已成为影响我国瓜类品质和产量的重要病害。本文从瓜类作物枯萎病的防治方法、致病机理和分子生物学研究等方面综述其研究现状,并对今后的研究方向进行展望。