Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Avian pluripotent stem cells

Pluripotent embryonic stem cells are undifferentiated cells capable of proliferation and self-renewal and have the capacity to differentiate into all somatic cell types and the germ line. They provide an in vitro model of early embryonic differentiation and are a useful means for targeted manipulation of the genome. Pluripotent stem cells in the chick have been derived from stage X blastoderms and 5.5 day gonadal primordial germ cells (PGCs). Blastoderm-derived embryonic stem cells (ESCs) have the capacity for in vitro differentiation into embryoid bodies and derivatives of the three primary germ layers. When grafted onto the chorioallantoic membrane, the ESCs formed a variety of differentiated cell types and attempted to organize into complex structures. In addition, when injected into the unincubated stage X blastoderm, the ESCs can be found in numerous somatic tissues and the germ line. The potential give rise to somatic and germ line chimeras is highly dependent upon the culture conditions and decreases with passage. Likewise, PGC-derived embryonic germ cells (EGCs) can give rise to simple embryoid bodies and can undergo some differentiation in vitro. Interestingly, chicken EG cells contribute to somatic lineages when injected into the stage X blastoderm, but only germ line chimeras have resulted from EGCs injected into the vasculature of the stage 16 embryo. To date, no lines of transgenic chickens have been generated using ESCs or EGCs. Nevertheless, progress towards the culture of avian pluripotent stem cells has been significant. In the future, the answers to fundamental questions regarding segregation of the avian germ line and the molecular basis of pluripotency should foster the full use of avian pluripotent stem cells.     

ARID3B Directly Regulates Ovarian Cancer Promoting Genes

The DNA-binding protein AT-Rich Interactive Domain 3B (ARID3B) is elevated in ovarian cancer and increases tumor growth in a xenograft model of ovarian cancer. However, relatively little is known about ARID3B''s function. In this study we perform the first genome wide screen for ARID3B direct target genes and ARID3B regulated pathways. We identified and confirmed numerous ARID3B target genes by chromatin immunoprecipitation (ChIP) followed by microarray and quantitative RT-PCR. Using motif-finding algorithms, we characterized a binding site for ARID3B, which is similar to the previously known site for the ARID3B paralogue ARID3A. Functionality of this predicted site was demonstrated by ChIP analysis. We next demonstrated that ARID3B induces expression of its targets in ovarian cancer cell lines. We validated that ARID3B binds to an epidermal growth factor receptor (EGFR) enhancer and increases mRNA expression. ARID3B also binds to the promoter of Wnt5A and its receptor FZD5. FZD5 is highly expressed in ovarian cancer cell lines, and is upregulated by exogenous ARID3B. Both ARID3B and FZD5 expression increase adhesion to extracellular matrix (ECM) components including collagen IV, fibronectin and vitronectin. ARID3B-increased adhesion to collagens II and IV require FZD5. This study directly demonstrates that ARID3B binds target genes in a sequence-specific manner, resulting in increased gene expression. Furthermore, our data indicate that ARID3B regulation of direct target genes in the Wnt pathway promotes adhesion of ovarian cancer cells.     

Glymphatic distribution of CSF-derived apoE into brain is isoform specific and suppressed during sleep deprivation

Background

Apolipoprotein E (apoE) is a major carrier of cholesterol and essential for synaptic plasticity. In brain, it’s expressed by many cells but highly expressed by the choroid plexus and the predominant apolipoprotein in cerebrospinal fluid (CSF). The role of apoE in the CSF is unclear. Recently, the glymphatic system was described as a clearance system whereby CSF and ISF (interstitial fluid) is exchanged via the peri-arterial space and convective flow of ISF clearance is mediated by aquaporin 4 (AQP4), a water channel. We reasoned that this system also serves to distribute essential molecules in CSF into brain. The aim was to establish whether apoE in CSF, secreted by the choroid plexus, is distributed into brain, and whether this distribution pattern was altered by sleep deprivation.

Methods

We used fluorescently labeled lipidated apoE isoforms, lenti-apoE3 delivered to the choroid plexus, immunohistochemistry to map apoE brain distribution, immunolabeled cells and proteins in brain, Western blot analysis and ELISA to determine apoE levels and radiolabeled molecules to quantify CSF inflow into brain and brain clearance in mice. Data were statistically analyzed using ANOVA or Student’s t- test.

Results

We show that the glymphatic fluid transporting system contributes to the delivery of choroid plexus/CSF-derived human apoE to neurons. CSF-delivered human apoE entered brain via the perivascular space of penetrating arteries and flows radially around arteries, but not veins, in an isoform specific manner (apoE2?>?apoE3?>?apoE4). Flow of apoE around arteries was facilitated by AQP4, a characteristic feature of the glymphatic system. ApoE3, delivered by lentivirus to the choroid plexus and ependymal layer but not to the parenchymal cells, was present in the CSF, penetrating arteries and neurons. The inflow of CSF, which contains apoE, into brain and its clearance from the interstitium were severely suppressed by sleep deprivation compared to the sleep state.

Conclusions

Thus, choroid plexus/CSF provides an additional source of apoE and the glymphatic fluid transporting system delivers it to brain via the periarterial space. By implication, failure in this essential physiological role of the glymphatic fluid flow and ISF clearance may also contribute to apoE isoform-specific disorders in the long term.

     

Deciphering the SUMO code in the kidney

SUMOylation of proteins is an important regulatory element in modulating protein function and has been implicated in the pathogenesis of numerous human diseases such as cancers, neurodegenerative diseases, brain injuries, diabetes, and familial dilated cardiomyopathy. Growing evidence has pointed to a significant role of SUMO in kidney diseases such as DN, RCC, nephritis, AKI, hypertonic stress and nephrolithiasis. Recently, emerging studies in podocytes demonstrated that SUMO might have a protective role against podocyte apoptosis. However, the SUMO code responsible for beneficial outcome in the kidney remains to be decrypted. Our recent experiments have revealed that the expression of both SUMO and SUMOylated proteins is appreciably elevated in hypoxia‐induced tubular epithelial cells (TECs) as well as in the unilateral ureteric obstruction (UUO) mouse model, suggesting a role of SUMO in TECs injury and renal fibrosis. In this review, we attempt to decipher the SUMO code in the development of kidney diseases by summarizing the defined function of SUMO and looking forward to the potential role of SUMO in kidney diseases, especially in the pathology of renal fibrosis and CKD, with the goal of developing strategies that maximize correct interpretation in clinical therapy and prognosis.     

Role of IKK/NF-κB signaling in extinction of conditioned place aversion memory in rats

The inhibitor κB protein kinase/nuclear factor κB (IKK/NF-κB) signaling pathway is critical for synaptic plasticity. However, the role of IKK/NF-κB in drug withdrawal-associated conditioned place aversion (CPA) memory is unknown. Here, we showed that inhibition of IKK/NF-κB by sulphasalazine (SSZ; 10 mM, i.c.v.) selectively blocked the extinction but not acquisition or expression of morphine-induced CPA in rats. The blockade of CPA extinction induced by SSZ was abolished by sodium butyrate, an inhibitor of histone deacetylase. Thus, the IKK/NF-κB signaling pathway might play a critical role in the extinction of morphine-induced CPA in rats and might be a potential pharmacotherapy target for opiate addiction.     

三种不同方法固定的石蜡切片中RNA的分析

研究在 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定的石蜡切片中提取RNA的质量和数量 .取 2 5 0g体重的Wistar大鼠的肾脏 ,分别采用 10 %中性福尔马林、丙酮、甲醇 氯仿 冰醋酸 3种方法固定 ,石蜡包埋 ,H E染色 ;采用RNA裂解液、TRIZOL试剂 2种方法提取切片RNA ,逆转录为cDNA ,采用普通PCR和SYBRGREEN 1定量PCR分析RNA质量和数量 .结果表明 ,3种固定方法都可保持组织良好的结构和形态 ;采用 2种提取方法 ,均可经RT PCR扩增出 180bp大鼠磷酸甘油醛脱氢酶 (G3PDH)、5 6 5bpβ肌动蛋白 (β actin)、10 0bp纤溶酶系活化剂抑制物 1(PAI 1) ;但采用RNA裂解液时 ,比TRIZOL试剂可提取更多的RNA .