沈阳地区北虫草野生与市售菌株人工培育子实体的蛋白质组学比较

【背景】北虫草作为冬虫夏草的代用品,具有与冬虫夏草类似的药理活性,其富含的蛋白质和氨基酸通常作为衡量真菌营养价值的重要指标,从中分离纯化具有潜在临床应用价值的蛋白质或多肽,已成为一个研究热点。【目的】检测沈阳北虫草野生与市售菌株人工培育子实体的蛋白质组成,分析相同培育条件下获得的蛋白种类、数量及其功能的差异,为深入研究鉴定沈阳地区北虫草药用蛋白和针对性驯化提供了蛋白质组学数据基础。【方法】采集沈阳棋盘山野生北虫草菌株,与市售人工栽培北虫草菌株同期分别经组织分离、液体发酵后培育获得子实体,通过蛋白提取、胰酶酶解后,采用非标定量技术液相色谱-质谱联用方法,对野生和市售来源培育的子实体样本进行定量蛋白组的研究。【结果】共鉴定到9 233条特异性肽段和1 923个蛋白,其中含有1 163个可定量蛋白,野生来源培育子实体有214个蛋白表达发生上调,181个蛋白表达发生下调,对这些差异蛋白进行功能富集分析发现,其主要参与能量生产/转换、氨基酸转运/代谢、抗氧化功能。在相同的营养摄取条件下,野生来源培育菌种在各个能量代谢、氨基酸代谢功能中的相关蛋白表达量高于市售来源培育的菌种。野生来源培育菌种的一种抗氧化重要蛋白(Gene Name:ISF_02112)表达量远远高于(Fold Change9)市售来源培育菌种。同时与抗氧化和代谢功能相关的差异蛋白有22个。【结论】沈阳地区北虫草野生菌株经适当人工培育会保留部分优良的生物学特性,2种来源菌株培育的子实体具有丰富及优异抗氧化功能的蛋白,子实体蛋白的抗氧化能力与其整体代谢能力相关。本研究结果为深入研究鉴定北虫草药用蛋白和针对性驯化提供蛋白质组学数据基础。     

Genotypic analysis of degenerative Cordyceps militaris cultured in the pupa of Bombyx mori

The chemical composition and pharmacological effects of Cordyceps militaris are similar to those of Cordyceps sinensis, with the former undergoing greater development and utilization. Strain degeneration is a common phenomenon that occurs with high frequency during the subculturing of C. militaris, however, and the mechanism underlying strain degeneration remains unclear. In this study, we used touch‐down PCR to compare the ITS1 + 5.8S + ITS2, 18S, 28S and mating‐type (MAT) regions sequence of wild‐type and degenerated strains of C. militaris. We also used quantitative real‐time PCR to analyze expression levels of the CmMAT gene. Sequence analysis showed that the ITS1 + 5.8S + ITS2 and 28S regions of degenerated and wild‐type strains were completely identical, the 18S region of the degenerated strain contained seven single‐base mutations, including six base substitutions and one single‐base insertion. Compared with the wild‐type strain, the degenerated strain contained a deletion of the MAT1–2‐1 region, three base substitutions in the MAT1–1‐1 region, and a base substitution in the MAT1–1‐2 region that causes a glycine‐to‐valine amino acid substitution. Quantitative real‐time PCR analysis detected no CmMAT1–2‐1 gene expression in the degenerated strain, confirming the deletion of the CmMAT1–2‐1 gene. Expression levels of the CmMAT1–1‐1 and CmMAT1–1‐2 genes were significantly down‐regulated to only 7.5 % and 4.4 %, respectively, that of the wild‐type strain. These results indicate that 18S and MAT region mutations, as well as down‐regulated of CmMAT gene expression levels, may play important roles in C. militaris degeneration. This study provides a theoretical basis for further elucidation of the molecular mechanisms of C. militaris degeneration.     

Molecular cloning and sequence analysis of the β-1,3-glucan synthase catalytic subunit gene from a medicinal fungus, Cordyceps militaris

The entomopathogenic fungus Cordyceps militaris belongs to vegetable wasps and plant worms and is used as herbal medicine, but β-1,3-glucan biosynthesis has been poorly studied in C. militaris. The fungal FKS1 gene encodes an integral membrane protein that is the catalytic subunit of β-1,3-glucan synthase. Here, we isolated cDNA clones encoding a full-length open reading frame of C. militaris FKS1. Cordyceps militaris Fks1 protein is a 1981 amino acid protein that shows significant similarity with other fungal Fks proteins. This study is the first report of molecular cloning of the β-1,3-glucan synthase catalytic subunit gene from vegetable wasps and plant worms.     

Expression of EGR-1 in a subset of olfactory bulb dopaminergic cells

In the adrenal medulla, binding of the immediate early gene (IEG) proteins, EGR-1 (ZIF-268/KROX-24/NGFI-A) and AP-1, to the tyrosine hydroxylase (Th) proximal promoter mediate inducible Th expression. The current study investigated the potential role of EGR-1 in inducible Th expression in the olfactory bulb (OB) since IEGs bound to the AP-1 site in the Th proximal promoter are also necessary for activity-dependent OB TH expression. Immunohistochemical analysis of a naris-occluded mouse model of odor deprivation revealed weak EGR-1 expression levels in the OB glomerular layer that were activity-dependent. Immunofluorescence analysis indicated that a majority of glomerular cells expressing EGR-1 also co-expressed TH, but only small subset of TH-expressing cells contained EGR-1. By contrast, granule cells, which lack TH, exhibited EGR-1 expression levels that were unchanged by naris closure. Together, these finding suggest that EGR-1 mediates activity-dependent TH expression in a subset of OB dopaminergic neurons, and that there is differential regulation of EGR-1 in periglomerular and granule cells.     

Phosphatidylinositol Transfer Protein Expression Altered by Aging and Parkinson Disease

1. Phosphatidylinositol transfer proteins (PI-TP) are responsible for the transport of phosphatidylinositol (PI) and other phospholipids from endoplasmic reticulum to the other membranes and indirectly for lipid mediated signaling. Till now little is known about PI-TPs in brain aging and neurodegeneration. The aim of this study was to investigate expression of PI-TP in the brain during aging and in animal's model of Parkinson disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Moreover, in vitro, effect of 1-methyl-4-phenyl-pyridine cation (MPP⁺) on PI-TP, tyrosine hydroxylase (TH) protein level, and viability of cells was investigated.2. Wistar rats 4, 24, and 36 months old and C57/BL mice and rat pheochromocytoma (PC12) cell line were used for the studies. Mice C57/BL received three injections of MPTP in saline at 2 h intervals in a total dose of 40 mg/kg and then after 3, 7, and 14 days they were used for the investigation. PC12 cells were treated with increasing concentration (50–300 μM) of MPP⁺ for 24 h at 37°C. The level of PI-TP_{α and β} and TH were determined using Western Blot analysis.3. Our data indicated that PI-TP_{α and β} level decreased in brain of 36 months old rat by 20% comparing to the control value (4 months old). In animal's model of PD, PI-TP_{α and β} level was significantly lower by 85, 69, 64% in striatum at 3, 7, and 14 days after MPTP injection, respectively, compared to the control value. MPP⁺ decreased PI-TP_{α and β}, TH expression, and viability of PC12 cells in a dose-dependent manner. H₂O₂, menadione, and NO donor significantly decreased the PI-TP level and viability of PC12 cells.4. Our results indicate the lower protein expression of PI-TP_{α and β} in aged brain and in PD and suggest that oxidative stress may be responsible for the alteration of PI-TP.     

Comparative studies of PC12 and mouse pheochromocytoma-derived rodent cell lines as models for the study of neuroendocrine systems

Summary We have compared PC12 cell lines derived from different laboratories and the newly developed mouse pheochromocytoma (MPC) cell line. Morphologically, there were distinct differences in size, shape, adherence, and clumping behavior, which varied in response to different culture media, growth substrates, and nerve growth factor. Quantitative messenger ribonucleic acid (mRNA) analysis showed significant variability in the expression of the catecholaminergic biosynthetic enzymes tyrosine hydroxylase (TH) phenylethanolamine N-methyltransferase (PNMT), the noradrenaline transporter (NAT), and neuron-specific enolase (NSE) between all lines examined. Of most significance were the increased levels of PNMT mRNA in the MPC cells, which were to 15-fold greater than in the PC12 cell lines grown under the same conditions in Dulbecco modified Eagle medium (P<0.05). Growth of MPC cells in Roswell Park Memorial Institute media induced a further significant increase in PNMT gene expression (P≤0.05). Immunohistochemistry for TH, PNMT. and NAT was generally consistent with mRNA analysis, with the MPC cells demonstrating strong immunoreactivity, for PNMT. The MPC cells showed the highest levels of desipramine-sensitive [³H] noradrenaline uptake activity (threefold > than PC12 American Type Culture Center line, P≤0.05), despite relatively low levels of NAT mRNA. These results indicate that PC12 cell lines should be carefully chosen for optimal utility in the study of chromaffin cell or sympathetic neuron biology and that cell features will be influenced by type of media and substrate chosen. Furthermore, they confirm that the new MPC cell line is likely a useful model for the study of adrenergic mechanisms or studies involving NAT.     

蛹虫草线粒体DNA与细胞核DNA进化关系的比较

【目的】分析蛹虫草是否存在核内线粒体DNA片段,比较蛹虫草线粒体DNA与细胞核DNA的碱基变异程度及所反映的菌株间的系统发育关系。【方法】通过本地BLAST或LAST对蛹虫草线粒体基因组和核基因组进行序列相似性搜索;从10个已知线粒体基因组的蛹虫草菌株中分别扩增7个细胞核蛋白编码基因片段,并与其在14个线粒体蛋白编码基因上的碱基变异情况进行比较。【结果】蛹虫草核基因组中存在5处较短的核内线粒体DNA片段,总长只有278bp。蛹虫草核DNA的变异频率整体上高于线粒体DNA。核DNA和线粒体DNA所反映的蛹虫草菌株间的系统发育关系存在显著差异。【结论】蛹虫草线粒体DNA与核DNA间不存在长片段的基因交流,二者变异频率不同,所反映的蛹虫草菌株间的系统发育关系也有差异。本研究增加了对蛹虫草线粒体与细胞核DNA进化关系的认识。     

The Use of Epstein–Barr Virus-Based Shuttle Vectors in Rat PC12 Cells

1. The rat pheochromocytoma PC12 cell line has been a commonly used model for studies of neuronal development, function, and death. Thus the abilityto transfect PC12 cells in an efficient manner and to manipulate their gene expression would enhance the usefulness of these cells.2. We demonstrate that EBV-based vectors provide a useful expression system for gene manipulation in rat PC12 cells.3. The EBV-based vectors replicate episomally in PC12 cells for at least 2months, as evidence by their recovery from the transfected cells and by the digestion of the episomal plasmid with the isoschizomer MboI and DpnI restriction enzymes.3. PC12 cells are efficiently transfected by EBV-based vectors both transiently and stably.4. Transfection of PC12 cells with an EBV-based vector containing tau cDNA in the antisense orientation resulted in a decrease in the level of tau protein in the transfected cells.5. The results demonstrate that EBV-based vectors can be a useful expression system for gene manipulation in PC12 cells.     

Targeted Gene Deletion in <Emphasis Type="Italic">Cordyceps militaris</Emphasis> Using the Split-Marker Approach

The macrofungus Cordyceps militaris contains many kinds of bioactive ingredients that are regulated by functional genes, but the functions of many genes in C. militaris are still unknown. In this study, to improve the frequency of homologous integration, a genetic transformation system based on a split-marker approach was developed for the first time in C. militaris to knock out a gene encoding a terpenoid synthase (Tns). The linear and split-marker deletion cassettes were constructed and introduced into C. militaris protoplasts by PEG-mediated transformation. The transformation of split-marker fragments resulted in a higher efficiency of targeted gene disruption than the transformation of linear deletion cassettes did. The color phenotype of the Tns gene deletion mutants was different from that of wild-type C. militaris. Moreover, a PEG-mediated protoplast transformation system was established, and stable genetic transformants were obtained. This method of targeted gene deletion represents an important tool for investigating the role of C. militaris genes.     

Region-specific effects of hypothyroidism on the relative expression of thyroid hormone receptors in adult rat brain

The aim of this study was to determine whether changes in the circulating thyroid hormone (TH) and brain synaptosomal TH content affected the relative levels of mRNA encoding different thyroid hormone receptor (TR) isoforms in adult rat brain. Northern analysis of polyA⁺RNA from cerebral cortex, hippocampus and cerebellum of control and hypothyroid adult rats was performed in order to determine the relative expression of all TR isoforms. Circulating and synaptosomal TH concentrations were determined by radioimmunoassay. Region-specific quantitative differences in the expression pattern of all TR isoforms in euthyroid animals and hypothyroid animals were recorded. In hypothyroidism, the levels of TRα2 mRNA (non-T₃-binding isoform) were decreased in all brain regions examined. In contrast the relative expression of TRα1 was increased in cerebral cortex and hippocampus, whereas in cerebellum remained unaffected. The TRβ1 relative expression in cerebral cortex and hippocampus of hypothyroid animals was not affected, whereas this TR isoform was not detectable in cerebellum. The TR isoform mRNA levels returned to control values following T₄ intraperitoneal administration to the hypothyroid rats. The obtained results show that in vivo depletion of TH regulates TR gene expression in adult rat brain in a region-specific manner. (Mol Cell Biochem 278: 93–100, 2005)     

Cytotoxic compounds against cancer cells from Bombyx mori inoculated with Cordyceps militaris

Two compounds, 3′-deoxyinosine and cordycepin, were isolated from Bombyx mori inoculated with Cordyceps militaris. In the bioassay examining cytotoxicity against cancer cells, both compounds showed toxicity against A549, PANC-1, and MCF-7 cancer cells.     

The Role of 3-<Emphasis Type="Italic">O</Emphasis>-Methyldopa in the Side Effects of <Emphasis Type="SmallCaps">l</Emphasis>-dopa

Long-term treatment of l-dopa for Parkinson’s disease (PD) patients induces adverse effects, including dyskinesia, on–off and wearing-off symptoms. However, the cause of these side effects has not been established to date. In the present study, therefore, 3-O-methyldopa (3-OMD), which is a major metabolite of l-dopa, was tested to determine whether it plays a role in the aforementioned adverse effects. The effects of 3-OMD on the dopaminergic nervous system in the brain were investigated, by examining behavioral, biochemical, and cellular changes in male Sprague–Dawley rats and catecholamine-producing PC12 neuronal cells. The results revealed that the intracerebroventricular (icv) injection of 1 μmol of 3-OMD impaired locomotor activities by decreasing movement time (MT), total distance (TD), and the number of movement (NM) by 70, 74 and 61%, respectively. The biochemical analysis results showed that a single administration of 1 μmole of 3-OMD decreased the dopamine turnover rate (DOPAC/DA) by 40.0% in the rat striatum. 3-OMD inhibited dopamine transporter and uptake in rat brain striatal membranes and PC12 cells. The subacute administration of 3-OMD (5 days, icv) also significantly impaired the locomotor activities and catecholamine levels. 3-OMD induced cytotoxic effects via oxidative stress and decreased mitochondrial membrane potential in PC12 cells, indicating that 3-OMD can damage neuronal cells. Furthermore, 3-OMD potentiated l-dopa toxicity and these toxic effects induced by both 3-OMD and l-dopa were blocked by vitamin E (α-tocopherol) in PC12 cells, indicating that 3-OMD may increase the toxic effects of l-dopa to some extent by oxidative stress. Therefore, the present study reveals that 3-OMD accumulation from long-term l-dopa treatment may be involved in the adverse effects of l-dopa therapy. Moreover, l-dopa treatment might accelerate the progression of PD, at least in part, by 3-OMD.     

Sequence analysis and heterologous expression of lectin-like gene CMLec3 from the medicinal fungus Cordyceps militaris

Through data mining of the Cordyceps militaris genome, a lectin-like encoding gene, CMLec3, was identified. In this study, the CMLec3 sequence was analyzed using bioinformatics approaches, and the gene was heterologously expressed in Escherichia coli BL21?cells. The biological activity of the product was examined. In addition, CMLec3 gene expression levels were assessed. The results showed that the CMLec3 protein contained a lectin domain structure and was successfully expressed. The CMLec3 protein partly inhibited HeLa cell proliferation. CMLec3 exhibited the highest gene expression in the primordium at a level 5.19 times that of the mycelium and 1.35 times that of the fruiting body. This suggests that the gene may be related to fruiting body development.     

Role of the HIF-1α/Nur77 axis in the regulation of the tyrosine hydroxylase expression by insulin in PC12 cells

Tyrosine hydroxylase (TH), catalyzing the conversion of tyrosine into l -DOPA, is the rate-limiting enzyme in dopamine synthesis. Defects in insulin action contribute to alterations of TH expression and/or activity in the brain and insulin increases TH levels in 1-methyl-4-phenylpyridinium (MPP+)-treated neuronal cells. However, the molecular mechanisms underlying the regulation of TH by insulin have not been elucidated yet. Using PC12 cells, we show for the first time that insulin increases TH expression in a biphasic manner, with a transient peak at 2 hr and a delayed response at 16 hr, which persists for up to 24 hr. The use of a dominant negative hypoxia-inducible factor 1-alpha (HIF-1α) and its pharmacological inhibitor chetomin, together with chromatin immunoprecipitation (ChIP) experiments for the specific binding to TH promoter, demonstrate the direct role of HIF-1α in the early phase. Moreover, ChIP experiments and transfection of a dominant negative of the nerve growth factor IB (Nur77) indicate the involvement of Nur77 in the late phase insulin response, which is mediated by HIF-1α. In conclusion, the present study shows that insulin regulates TH expression through HIF-1α and Nur77 in PC12 cells, supporting the critical role of insulin signaling in maintaining an appropriate dopaminergic tone by regulating TH expression in the central nervous system.     

蛹虫草饲料添加剂的研究现状及展望

蛹虫草饲料添加剂包括蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养残基提取物、蛹虫草菌固液发酵产物、微生物发酵蛹虫草残基等产品.蛹虫草饲料添加剂含有粗蛋白、粗脂肪、氨基酸等营养成分,以及虫草素、腺苷、多糖等活性成分,在畜禽、反刍动物、水产品等动物养殖中的应用均获得较好的效果.对蛹虫草子实体、蛹虫草培养残基、蛹虫草及其培养...