Identification of genes induced in alkaloid-producing cultures of Claviceps sp.

In order to identify genes which are expressed during alkaloid synthesis in an axenic culture of Claviceps sp. (strain ATCC 26245), a cDNA library from a producing culture was differentially screened with cDNA from producing (cDNA+) and non-producing (cDNA–) cultures, respectively. Altogether, ten cDNA clones were obtained, the alkaloid-synthesis-correlated expression of which was confirmed by Northern analyses. Evaluation of their nucleotide and derived amino-acid sequences identified one gene unequivocally, coding for dimethylallyltryptophan-synthase (DMAT-S), the initial enzyme of the specific alkaloid pathway. For two other genes significant homologies to known fungal genes were detected: one clone showed homology to the Neurospora crassa ccg1 gene, coding for a clock-regulated putative general stress protein; seven cDNA clones, derived from the same gene, which is highly expressed under these conditions, contained typical hydrophobin domains and long stretches of asparagine/glycine repeats (like QID3 from Trichoderma harzianum), thus probably representing a cell-wall constituent. These data show that this is not only a successful approach to clone genes specific for the alkaloid-pathway of C. purpurea, but also of genes which might be involved in the differentiation of sclerotial hyphae, the prerequisite for alkaloid synthesis. Received: 22 November 1996     

Presenilin regulates extracellular regulated kinase (Erk) activity by a protein kinase C alpha dependent mechanism

Presenilin (PS1 and PS2) mutations cause early-onset familial Alzheimer's disease (AD). In addition to affecting β-amyloid precursor protein (APP) processing and Aβ generation, PSs regulate a number of signaling pathways. We previously showed that PSs regulate both phospholipase C (PLC) and protein kinase C (PKC) α and γ activities. We also reported that PS double knockout mouse embryonic fibroblasts (MEFs) have reduced levels of PKCα and enhanced levels of PKCδ. Here, we determined whether the PS modulation of PLC/PKC has consequences for extracellular regulated kinase (Erk) signaling. Erk has been suggested to be important in AD pathology by modulating APP processing and tau phosphorylation. We found that knocking out PS1 or PS2 alone resulted in increased Erk activity and that this effect could be reversed by the PKCα inhibitor Gö6976. We also found that Erk activity following either PLC or PKC stimulation was significantly lower in PS double knockout cells and that treatment with the PKC activator phorbol 12,13-dibutyrate (PdBu) down-regulated total-Erk levels in all cells except PS double knockouts. These results demonstrate that PSs regulate Erk activity through a PKCα dependent pathway and that disruption of PLC/PKC signaling in the absence of both PS1 and PS2 results in lower downstream activation of Erk.     

Altered chromatin landscape in circulating T follicular helper and regulatory cells following grass pollen subcutaneous and sublingual immunotherapy

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EB病毒LMP2A特异性CTL的体外诱导及分析

用EBV LMP2A重组痘苗病毒 (rVV LMP2A )转染人树突状细胞 (DC ) ,转染后的DC分别在第 1、 7、 14天刺激相同MHC背景的T细胞 ,在IL 2作用下诱导LMP2A特异性CTL。用LDH释放法检测CTL杀伤活性 ;流式细胞术 (FACS )检测CTL诱导分化过程中CD3+ 、CD4 + 、CD8+ 、CD5 6 + 等细胞的分群变化 ;RT PCR检测细胞分化过程中FasLmRNA表达 ;生物活性法检测功能性细胞因子IFN γ的分泌。结果显示本法诱导的CTL对靶细胞有特异性杀伤活性 ,第 2次和第 3次DC刺激后杀伤活性有所上升 ;在CTL诱导分化的第 7、 14、 2 1天细胞分群以CD4 + 、CD8+ 细胞为主 ;RT PCR证实所诱导的细胞内有FasLmRNA的表达 ;随细胞培养天数的增加IFN γ分泌增加 ,在第 14天达到较高水平。研究表明重组痘苗病毒载体rVV LMP2A转染的DC刺激T细胞可诱导出EBV LMP2A特异性CTL。     

神经激肽A在大鼠结肠发育中的定性定量分析

探讨神经激肽A(NKA)在大鼠结肠发育中的表达及变化,应用免疫组织化学PAP法和图像分析系统观测大鼠胚胎13d至成年结肠中NKA的表达情况。结果显示:(1)在结肠,首先于胚胎17 d的肌间丛及上皮处出现NKA-IR的表达,随发育相继出现于纵肌、环肌、粘膜肌、固有膜、粘膜下层和粘膜丛,30 d时具备成年分布特征;(2)定量分析的结果与NKA~IR在结肠各层的变化一致;(3)NKA-IR细胞具有典型的肠道内分泌细胞的形态特征和分布特征。结果提示:(1)NKA在结肠的发生发育主要在生前5 d~生后4周内;(2)NKA在结肠的发生发育有两个关键时期,即生前5 d~生后1周和生后1月末;(3)NKA-IR细胞可能是肠道内分泌NKA的内分泌细胞;(4)NKA可能对大鼠结肠的发育具有重要作用,可能与结肠功能的建立密切相关。     

cGMP对脑缺血再灌流后海马区星形胶质细胞活化的作用机制研究

为了观察 c GMP对海马区胶质纤维酸性蛋白合成调节的作用 ,本研究用钳夹沙土鼠的双侧颈总动脉制造脑缺血模型 ,进行了免疫荧光法染色。结果表明 :脑缺血再灌流后海马区 c GMP合成增加 ,多数 c GMP阳性细胞为星形胶质细胞 ,此细胞的多数呈 c GMP强阳性染色 ,胶质纤维酸性蛋白反应也多呈强阳性 ;使用鸟苷酸环化酶 ( GC)抑制剂 ODQ,则 c GMP合成减少 ,c GMP阳性细胞多呈弱阳性 ,同一细胞的胶质纤维酸性蛋白反应也多呈弱阳性。本实验结果提示 :c GMP可能与海马胶质纤维酸性蛋白的合成调节有关     

Mixed neuronal–glial tumor of the digestive tract: Distinctive entity from gastrointestinal stromal tumor?

A 53-year-old-woman presenting with pelvic discomfort was found to have a 9.5 cm tumor located in the wall of the ileon. Light microscopy showed that the tumor was made of fascicles of plump spindle cells and bizarre epithelioid cells. A cuff of lymphoid cells was also present at the tumor margin. The tumor cells strongly expressed tau protein, neuron-specific enolase, protein green product 9.5 and glial fibrillary acid protein (GFAP), but did not show positive immunostaining for S-100 protein, CD34 or CD117. The tumor showed unequivocal ultrastructural evidence of neural differentiation. Skeinoid fibers were scattered throughout the tumor. This is the first mixed neuronal-glial tumor of the digestive tract to be described in the literature. Such histological and immunohistochemical features could be misinterpreted as features of digestive schwannoma. We suggest that this tumor is distinct from gastrointestinal stromal tumors in lacking CD34 and CD117 expression.     

Expression of the 40 kDa catecholamine regulated protein in the normal and injured rat retina

Catecholamine regulated protein 40 (CRP40) has been shown to be expressed in the central nervous system (CNS) of several mammalian species where it may function in a similar manner to members of the heat shock protein (HSP) family. Immunohistochemical and immunoblotting techniques were utilized to investigate whether CRP40 is expressed in normal rat retinas. In addition, changes in CRP40 expression were studied following optic nerve transection. The immunohistochemical results showed that CRP40 is expressed in the normal rat retina. The protein was found to be highly expressed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer plexiform layer (OPL). In addition, a low level of CRP40 was found in the inner plexiform layer (IPL), and in the inner segment layer (ISL). No expression was found in the outer nuclear layer (ONL) of normal rat retina. The immunoblotting results show that CRP40 expression decreased in a time-dependent fashion after the optic nerve transection. This decrease indicates that the expression of CRP40 is dependent on the neuron's normal physiological state and that it plays an important function in physiological and pathological conditions in the retina.