Cryptic organisation within an apparently irregular rostrocaudal distribution of interneurons in the embryonic zebrafish spinal cord

The molecules and mechanisms involved in patterning the dorsoventral axis of the developing vertebrate spinal cord have been investigated extensively and many are well known. Conversely, knowledge of mechanisms patterning cellular distributions along the rostrocaudal axis is relatively more restricted. Much is known about the rostrocaudal distribution of motoneurons and spinal cord cells derived from neural crest but there is little known about the rostrocaudal patterning of most of the other spinal cord neurons. Here we report data from our analyses of the distribution of dorsal longitudinal ascending (DoLA) interneurons in the developing zebrafish spinal cord. We show that, although apparently distributed irregularly, these cells have cryptic organisation. We present a novel cell-labelling technique that reveals that DoLA interneurons migrate rostrally along the dorsal longitudinal fasciculus of the spinal cord during development. This cell-labelling strategy may be useful for in vivo analysis of factors controlling neuron migration in the central nervous system. Additionally, we show that DoLA interneurons persist in the developing spinal cord for longer than previously reported. These findings illustrate the need to investigate factors and mechanisms that determine “irregular” patterns of cell distribution, particularly in the central nervous system but also in other tissues of developing embryos.     

昆虫卵黄蛋白及其激素调控的研究进展

卵黄蛋白的结构及其合成、摄取过程与激素的调控机理是目前昆虫生理学的研究热点之一。近几年,随着分子克隆技术、基因工程手段和生物信息学的发展,对卵黄蛋白基因的研究将为寻找害虫生物防治提供新途径。本文对昆虫卵黄蛋白及其激素调控进行了综述。为防治害虫再猖獗的发生和促进大量繁殖益虫提供重要的理论依据。     

The small heat shock protein 20 RSI2 interacts with and is required for stability and function of tomato resistance protein I‐2

Race‐specific disease resistance in plants depends on the presence of resistance (R) genes. Most R genes encode NB‐ARC‐LRR proteins that carry a C‐terminal leucine‐rich repeat (LRR). Of the few proteins found to interact with the LRR domain, most have proposed (co)chaperone activity. Here, we report the identification of RSI2 (Required for Stability of I‐2) as a protein that interacts with the LRR domain of the tomato R protein I‐2. RSI2 belongs to the family of small heat shock proteins (sHSPs or HSP20s). HSP20s are ATP‐independent chaperones that form oligomeric complexes with client proteins to prevent unfolding and subsequent aggregation. Silencing of RSI2‐related HSP20s in Nicotiana benthamiana compromised the hypersensitive response that is normally induced by auto‐active variants of I‐2 and Mi‐1, a second tomato R protein. As many HSP20s have chaperone properties, the involvement of RSI2 and other R protein (co)chaperones in I‐2 and Mi‐1 protein stability was examined. RSI2 silencing compromised the accumulation of full‐length I‐2 in planta, but did not affect Mi‐1 levels. Silencing of heat shock protein 90 (HSP90) and SGT1 led to an almost complete loss of full‐length I‐2 accumulation and a reduction in Mi‐1 protein levels. In contrast to SGT1 and HSP90, RSI2 silencing led to accumulation of I‐2 breakdown products. This difference suggests that RSI2 and HSP90/SGT1 chaperone the I‐2 protein using different molecular mechanisms. We conclude that I‐2 protein function requires RSI2, either through direct interaction with, and stabilization of I‐2 protein or by affecting signalling components involved in initiation of the hypersensitive response.     

Anti-CD20scFv/IgGFc/CD80/CD28/zeta转染人T淋巴细胞对原代CLL细胞的作用

目的:将已成功构建表达anti-CD20scFv/CD80/CD28/zeta转染人T淋巴细胞,体外观察该类细胞特异性清除CD20+原代慢性淋巴细胞白血病(CLL)细胞的能力,为肿瘤的过继免疫治疗提供新思路。方法:将本室成功构建的含anti-CD20scFv/IgGFc/CD80片段的PLNCX质粒,转染PA317包装细胞,挑取高滴度的包装细胞株收获逆转录病毒,用收获的病毒感染刺激分裂的人外周血T细胞,经G418筛选后与CD20+原代CLL细胞在体外共同培养,在显微镜下观察CD20+的原代CLL细胞生长状态,ELISA检测试剂盒检测T细胞分泌细胞因子的功能。结果:重组基因修饰的T细胞能在体外杀伤CD20+原代CLL细胞,而对CD20-细胞无杀伤作用;同时靶细胞为CD20+组上清液中IL-2(1301.00pg/ml)和IFN-γ(602.18pg/ml)水平与CD20-组相比明显升高。结论:嵌合锚定T细胞能够成功构建;该类T细胞在体外能特异性杀伤CD20+的原代CLL细胞。     

PURIFICATION AND CHARACTERIZATION OF THE 20S PROTEASOME FROM THEALGA CHARA CORALLINA (CHAROPHYCEAE)1

We purified the 20S proteasome from the alga Chara corallina Willd with DEAE–ion‐exchange column chromatography and preparative nondenaturing PAGE. The analysis of the purified enzyme bynondenaturing PAGE gave a single band whose molecular mass was estimated to be about 600,000 Da by gel permeation chromatography and whose isoelectric point was at pH 5.5. Two‐dimensional gel electrophoresis gave at least 12 spots with molecular masses from 26,000 to 32,000 Da in a wide range of isoelectric points. The 20S proteasome hydrolyzed three types of artificial substrates used to differentiate chymotrypsin‐like, trypsin‐like, and peptidyl glutamyl peptidase activities. Both the chymotrypsin‐like and the peptidyl glutamyl peptidase activities were enhanced by SDS. In the presence of 0.03% SDS, the optimal pH for both activities was 8.5. Trypsin‐like activity of the 20S proteasome had a broad pH optimum in an alkaline region and was not activated but inhibited by SDS. Its chymotrypsin‐like activity was inhibited by N‐ethylmaleimide, p‐chloromercuribenzoic acid, and chymostatin. In contrast, its peptidyl glutamyl peptidase activity was not inhibited by chymostatin. Moreover, proteasome inhibitors MG 115 and MG 135 were effective against the chymotrypsin‐like activity and less so against the peptidyl glutamyl peptidase activity. These properties were very similar to those of the proteasomes of mammalian, yeast, and spinach cells. The large size of Chara cells will make in vivo manipulations and investigations of the proteasome proteolytic system possible.     

CCR9 and CCL25: A review of their roles in tumor promotion

Chemokines constitute a superfamily of small chemotactic cytokines with functions that are based on interactions with their corresponding receptors. It has been found that, among other functions, chemokines regulate the migratory and invasive abilities of cancer cells. Multiple studies have confirmed that chemokine receptor 9 (CCR9) and its exclusive ligand, chemokine 25 (CCL25), are overexpressed in a variety of malignant tumors and are closely associated with tumor proliferation, apoptosis, invasion, migration and drug resistance. This review evaluates recent advances in understanding the role of CCR9/CCL25 in cancer development. First, we outline the general background of chemokines in cancer and the structure and function of CCR9 and CCL25. Next, we describe the basic function of CCR9/CCL25 in the cancer process. Then, we introduce the role of CCR9/CCL25 and related signaling pathways in various cancers. Finally, future research directions are proposed. In general, this paper is intended to serve as a comprehensive repository of information on this topic and is expected to contribute to the design of other research projects and future efforts to develop treatment strategies for ameliorating the effects of CCR9/CCL25 in cancer.     

Phosphorylation of SMURF2 by ATM exerts a negative feedback control of DNA damage response

Timely repair of DNA double-strand breaks (DSBs) is essential to maintaining genomic integrity and preventing illnesses induced by genetic abnormalities. We previously demonstrated that the E3 ubiquitin ligase SMURF2 plays a critical tumor suppressing role via its interaction with RNF20 (ring finger protein 20) in shaping chromatin landscape and preserving genomic stability. However, the mechanism that mobilizes SMURF2 in response to DNA damage remains unclear. Using biochemical approaches and MS analysis, we show that upon the onset of the DNA-damage response, SMURF2 becomes phosphorylated at Ser³⁸⁴ by ataxia telangiectasia mutated (ATM) serine/threonine kinase, and this phosphorylation is required for its interaction with RNF20. We demonstrate that a SMURF2 mutant with an S384A substitution has reduced capacity to ubiquitinate RNF20 while promoting Smad3 ubiquitination unabatedly. More importantly, mouse embryonic fibroblasts expressing the SMURF2 S384A mutant show a weakened ability to sustain the DSB response compared with those expressing WT SMURF2 following etoposide treatment. These data indicate that SMURF2-mediated RNF20 ubiquitination and degradation controlled by ataxia telangiectasia mutated–induced phosphorylation at Ser³⁸⁴ constitutes a negative feedback loop that regulates DSB repair.     

(20S)‐Protopanaxadiol Ginsenosides Induced Cytotoxicity via Blockade of Autophagic Flux in HGC‐27 Cells

(20S)‐Protopanaxadiol ginsenosides Rg3, Rh2 and PPD have been demonstrated for their anticancer activity. However, the underlying mechanism of their antitumor activity remains unclear. In the present study, we investigated the role of these three ginsenosides on cell proliferation and death of human gastric cancer cells (HGC‐27 cells). The sulforhodamine B (SRB) assay, Western blot analysis, fluorescence microscopy, confocal microscopy, high performance liquid chromatography (HPLC) analysis, flow cytometry, and transmission electron microscopy (TEM) were used to evaluate cell proliferation, apoptosis, and autophagy. The results showed that both Rh2 and PPD were more effective than Rg3 in inhibiting HGC‐27 cell proliferation and inducing cytoplasmic vacuolation, while no significant changes in apoptosis were observed. Interestingly, cytoplasmic vacuolation and blockade of autophagy flux were observed after treatment with Rh2 and PPD. Rh2 obviously up‐regulated the expression of the LC3II and p62. Furthermore, the increase in lysosomal pH and membrane rupture was observed in Rh2‐treated and PPD‐treated cells. When HGC‐27 cells were pretreated with bafilomycin A1, a specific inhibitor of endosomal acidification, cellular vacuolization was increased, and the cell viability was significantly decreased, which indicated that Rh2‐induced lysosome‐damage accelerated cell death. Furthermore, data derived from mitochondrial analysis showed that excessive mitochondrial reactive oxygen species (ROS) and dysregulation of mitochondrial energy metabolism were caused by Rh2 and PPD treatment in HGC‐27 cells. Taken together, these phenomena indicated that Rh2 and PPD inhibited HCG‐27 cells proliferation by inducing mitochondria damage, dysfunction of lysosomes, and blockade of autophagy flux. The number of glycosyl groups at C‐3 position could have an important effect on the cytotoxicity of Rg3, Rh2 and PPD.