Inframolecular studies of the protonation of adenophostin A: comparison with 1-D-myo-inositol 1,4,5-trisphosphate

Adenophostin A is a glyconucleotide natural product with the highest known potency for the D-myo-inositol 1,4,5-trisphosphate receptor. Using synthetic adenophostin A we have investigated the macroscopic and microscopic protonation process of this compound by performing (31)P NMR, (1)H NMR, and potentiometric titration experiments. The logarithms of the first to the fourth stepwise protonation constants are, respectively, log K(1) = 8.48, log K(2) = 6.20, log K(3) = 4.96, and log K(4) = 3.80. The latter constant refers to the protonation equilibrium involving the N1 adenine nitrogen. From the microconstants the protonation fractions of each individual phosphate group can be calculated. Remarkably, the ionization state of the phosphates of adenophostin A at near physiological pH is very similar to those of inositol 1,4,5-trisphosphate, indicating that differences in phosphate charge cannot account for the high potency of this molecule. The analysis of the (1)H chemical shifts vs pH provided complementary conformational information. In particular, a slight "wrongway shift" of H1" can be related to the protonation of P2, thus indicating a short H1"-P2 distance. Our results are in line with a recently published model in which, however, a certain degree of constraint would keep the ribose 2'-phosphate moiety close to the glucose ring phosphates.     

Corticotropin-releasing factor receptor 1 in mouse spleen: expression after immune stimulation and identification of receptor-bearing cells

A specific polyclonal Ab against the N-terminal domain of corticotropin-releasing factor (CRF) receptor, type 1 (CRF-R1), was employed to an immunohistochemical analysis of the spleen from naive mice and mice exposed to an immune challenge. Cell types stained with anti-CRF-R1 Ab were identified by their nuclear shapes and colocalization with the cell type-specific markers ER-MP58, ER-MP20, Moma-1, Moma 2, anti-CD3e mAbs, and anti-Ig Ab. Only a few clusters of CRF-R1+ cells were found in spleen sections of naive mice at sites typical for granulopoietic islands. However, a 17-fold increase in the mean number of CRF-R1+ cells was noted within hours following a challenge of acute systemic inflammation induced by i.p. administration of LPS. The majority of these cells were identified as mature neutrophils. CRF-R1 was shown to mediate suppression of the IL-1beta secretion by these cells. However, at later time points a large number of granulocyte-macrophage precursors was strongly labeled with anti-CRF-R1 Ab. Western blot analysis of splenic membranes from animals treated with LPS revealed a m.w. of approximately 70,000 for CRF-R1. Subcellular staining patterns were suggestive for the predominant localization of CRF-R1 on granule membranes. CRF-R1 mRNA was detected in spleen but not in bone marrow and peripheral blood leukocytes from naive mice. Thus, it was indicated that CRF-R1 was not produced constitutively by mature or immature neutrophils. Its production was rather triggered by inflammatory stimuli.     

Tumorigenic mutations in VHL disrupt folding in vivo by interfering with chaperonin binding

The eukaryotic chaperonin TRiC/CCT mediates folding of an essential subset of newly synthesized proteins, including the tumor suppressor VHL. Here we show that chaperonin binding is specified by two short hydrophobic beta strands in VHL that, upon folding, become buried within the native structure. These TRiC binding determinants are disrupted by tumor-causing point mutations that interfere with chaperonin association and lead to misfolding. Strikingly, while unable to fold correctly in vivo, some of these VHL mutants can reach the native state when refolded in a chaperonin-independent manner. The specificity of TRiC/CCT for extended hydrophobic beta strands may help explain its role in folding aggregation-prone polypeptides. Our findings reveal a class of disease-causing mutations that inactivate protein function by disrupting chaperone-mediated folding in vivo.     

Ionomycin-activated calpain triggers apoptosis. A probable role for Bcl-2 family members

Ubiquitous calpains (mu- and m-calpain) have been repeatedly implicated in apoptosis, but the underlying mechanism(s) remain(s) to be elucidated. We examined ionomycin-induced cell death in LCLC 103H cells, derived from a human large cell lung carcinoma. We detected hallmarks of apoptosis such as membrane blebbing, nuclear condensation, DNA ladder formation, caspase activation, and poly-(ADP-ribose)polymerase cleavage. Apoptosis was prevented by preincubation of the cells with the calpain inhibitor acetyl-calpastatin 27-peptide and the caspase inhibitor Z-DEVD-fmk, implicating both the calpains and caspases in the apoptotic process. The apoptotic events correlated in a calpastatin-inhibitable manner with Bid and Bcl-2 decrease and with activation of caspases-9, -3, and -7. In vitro both ubiquitous calpains cleaved recombinant Bcl-2, Bid, and Bcl-x(L) at single sites truncating their N-terminal regions. Binding studies revealed diminished interactions of calpain-truncated Bcl-2 and Bid with immobilized intact Bcl-2 family proteins. Moreover, calpain-cleaved Bcl-2 and Bid induced cytochrome c release from isolated mitochondria. We conclude that ionomycin-induced calpain activation promotes decrease of Bcl-2 proteins thereby triggering the intrinsic apoptotic pathway.     

光照对柑橘果皮类胡萝卜素和色泽形成的影响

以“红柿柑”为试材,在柑橘果实膨大末期通过套袋遮光处理以抑制果皮光合作用,研究光照对果皮糖、叶绿素、类胡萝卜素及果实外观色泽的影响。结果表明,遮光后果皮叶绿素含量迅速下降引起果实转色提早,但各种类胡萝卜素含量及总量并未提高,而是显著下降;至果实成熟时由于遮光与光照处理的果皮叶绿素均消失,遮光果实类胡萝卜素含量低颜色变淡,与光照处理相比,遮光前期果皮糖含量下降不大,而后期下降明显;若在后期去袋照光,果皮糖含量上升,与此相应,类胡萝卜素,尤其是β-隐黄质的积累增加,颜色加深,表明光对果皮类胡萝卜素合成尤其是β-隐黄质的积累有促进作用,其原因是光以环境信号的方式影响果皮的类胡萝卜素形成。     

In vivo kinetics of protein targeting to the endoplasmic reticulum determined by site-specific phosphorylation

We have developed a novel assay to detect the cytosolic localization of protein domains by inserting a short consensus sequence for phosphorylation by protein kinase A. In transfected COS-1 cells, this sequence was labeled efficiently with [(32)P]phosphate only when exposed to the cytosol and not when translocated into the lumen of the endoplasmic reticulum. The phosphorylation state of this sequence can therefore be used to determine the topology of membrane proteins. This assay is sufficiently sensitive to detect even the transient cytosolic exposure of the N-terminal domain of a membrane protein with a reverse signal-anchor sequence. The extent of phosphorylation per newly synthesized polypeptide was shown to reflect the time of exposure to the cytosol, which depends on translation, targeting and translocation of the N-terminus. By altering the length of the N-terminal domain or manipulating the translation rate, it was determined that protein targeting is rapid and requires only a few seconds. The rate of N-terminal translocation was estimated to be approximately 1.6 times the rate of translation.     

三种苏铁植物呈现出迥异的水力安全边界北大核心CSCD

为探究苏铁植物的水力安全边界(hydraulic safety margins,HSM),该试验选用经典的自然干燥法和最新发表的抽气法测定三种同质园苏铁植物抗旱性(即叶轴木质部脆弱性曲线),获得抗旱指标P_(50)和P_(88)(导水率丧失或气体抽取量分别为50%和88%时的木质部水势),与叶片正午水势计算HSM,结合管胞性状分析。结果表明:(1)苏铁(Cycas revoluta)、越南篦齿苏铁(C.elongata)、摩瑞大泽米苏铁(Macrozamia moorei)的管胞长度分别为(4413±378)、(5146±730)、(6954±862)μm,苏铁、越南篦齿苏铁与摩瑞大泽米苏铁差异显著(P<0.05)。(2)两种方法测定的脆弱性曲线都呈典型的“S”型,苏铁、越南篦齿苏铁、摩瑞大泽米苏铁的P_(50H)(导水率丧失50%时的木质部水势)和P_(50P)(气体抽取量为50%时的木质部水势)分别为-2.5、(-2.4±0.5)MPa,-2.3、(-3.6±0.7)MPa,-1.5、(-1.8±0.2)MPa,在已发表的裸子植物数值范围内。P_(50)和P_(88)具有显著一致性(除了越南篦齿苏铁的P_(50P)比P_(50H)更低,表示更强抗旱性),与已发表的其他木质部管胞物种通过水力学法和抽气法获得的P_(50)和P_(88)比较分析,具有显著相关性(R^(2)=0.72,P=0.0081;R^(2)=0.87,P=0.0007)。(3)自然干燥法和抽气法计算的HSMs具有相同的趋势,摩瑞大泽米苏铁为负值,而苏铁和越南篦齿苏铁为正值。综上所述,三种苏铁植物的抗旱性均在已发表的裸子植物范围内,两种方法都适于测定木质部管胞结构的苏铁类植物脆弱性曲线,苏铁、越南篦齿苏铁与摩瑞大泽米苏铁具有不同的水力安全边界。利用脆弱性曲线和正午水势探讨苏铁植物的水力安全边界,为苏铁植物的水分监测、管理和保育提供依据。     

基于微卫星引物鉴别不同地理种群玉米螟赤眼蜂和螟黄赤眼蜂

不同地理种群的赤眼蜂在遗传、生理和生态适应性等方面均表现出不同程度的分化。为探究不同地理种群的玉米螟赤眼蜂Trichogramma ostriniae和螟黄赤眼蜂Trichogramma chilonis在基因型上的差异并进行准确鉴定,本研究筛选了可用以区分不同地理种群玉米螟赤眼蜂和螟黄赤眼蜂的微卫星引物。结果表明：从已报道的10对微卫星引物中,筛选出2对引物（序列号：KT834825,KT834827）可以区分玉米螟赤眼蜂中的黑龙江地理种群与吉林和辽宁两个地理种群;并筛选出2对引物（序列号：KT834822,KT834825）可以区分黑龙江、贵州和广东3个不同地理种群的螟黄赤眼蜂。该结果进一步证实赤眼蜂不同地理种群间可能存在明显的基因型差异,研究结果为精准鉴别玉米螟赤眼蜂和螟黄赤眼蜂不同地理种群,以及进一步探寻优势种群的高效繁育与应用技术奠定了理论基础。     

朊病毒病治疗方法的研究进展

朊病毒是一种由体内正常朊蛋白转化形成的传染性蛋白质,朊病毒病是由朊病毒引发的致命性神经退行性疾病。目前临床虽然尚无治疗朊病毒病的方法,但是大量的研究者已从多个角度进行研究,并取得了一定进展。对近期有关传统化学药物、基因治疗方法、免疫学治疗方法和同源朊蛋白的朊病毒病治疗方法进行了综述,并重点分析了新型靶向细胞内信号通路药物以及有潜在利用价值的线粒体相关朊病毒胞内作用信号通路,旨在为朊病毒新的研究方向提供理论依据,从而促进朊病毒病治疗方法应用于临床。     

Accelerated and safe expansion of human mesenchymal stromal cells in animal serum-free medium for transplantation and regenerative medicine

Human bone marrow mesenchymal stromal cells (hMSC) are currently investigated for a variety of therapeutic applications. However, most expansion protocols still use fetal calf serum (FCS) as growth factor supplement which is a potential source of undesired xenogeneic pathogens. We established an expansion protocol for hMSC based on the use of GMP-produced basic medium LP02 supplemented with 5% of platelet lysate (PL) obtained from human thrombocyte concentrates. Compared to FCS-supplemented culture conditions, we found a significant increase in both colony forming unit-fibroblast (CFU-F) as well as cumulative cell numbers after expansion. This accelerated growth is optimized by pooling of at least 10 thrombocyte concentrates. A minimal requirement is the use of 5% of PL with an optimal platelet concentration of 1.5 x 10(9)/ml, and centrifugation of thawed lysate at high speed. Cells expanded by this protocol meet all criteria for mesenchymal stromal cells (MSCs), e.g. plastic adherence, spindle-shaped morphology, surface marker expression, lack of hematopoietic markers, and differentiation capability into three mesenchymal lineages. MSC at passage 6 were cytogenetically normal and retained their immune-privileged potential by suppressing allogeneic reaction of T-cells. Additionally, gene expression profiles show increased mRNA levels of genes involved in cell cycle and DNA replication and downregulation of developmental and differentiation genes, supporting the observation of increased MSC-expansion in PL-supplemented medium. In summary, we have established a GMP-compatible protocol for safe and accelerated expansion of hMSC to be used in cell and tissue therapy.