红缘拟层孔菌发酵工艺的研究及其升罐培养

为筛选出红缘拟层孔菌发酵的最佳条件,并用于升罐发酵,试验优选出5个基础培养基,再以发酵菌丝体的生物量为指标,确定红缘拟层孔菌发酵的最佳条件。进行升罐发酵,间隔取出少量发酵液进行显微观察并检测葡萄糖含量,描述出菌丝体的生长状态。结果表明:红缘拟层孔菌发酵的最佳条件为马铃薯200 g/L,葡萄糖20 g/L,磷酸二氢钾1 g/L,硫酸镁1.5 g/L,维生素B10.4 g/L。升罐发酵中,菌丝干质量在前60 h处于快速的增长状态,最大为6.890 1 g/L,而60 h之后菌丝的生长变缓慢,到84 h之后,菌丝自溶,菌丝的产量开始下降。菌丝的葡萄糖含量经血糖仪检测在60 h时最高,达到0.296 5 g/L,之后呈现下降趋势。红缘拟层孔菌升罐发酵的最佳发酵时间为56~64 h。     

红缘拟层孔菌固体发酵产物体内抗肿瘤和抗氧化作用研究

研究了红缘拟层孔菌固体发酵产物对H22荷瘤小鼠的抗肿瘤和抗氧化作用,以抑瘤率、脾和胸腺指数、白细胞介素-2、干扰素-r、血管内皮细胞生长因子、超氧化物歧化酶、丙二醛、过氧化氢酶、谷胱甘肽过氧化物等指标来考察红缘拟层孔菌固体发酵产物对H22荷瘤小鼠肿瘤抑制和体内抗氧化作用。结果表明,固体发酵产物高剂量和中剂量组的抑瘤率分别为66.66%和64.70%,与阴性组比较,有显著的抗肿瘤作用（P<0.01）,HE染色切片也能观察到固体发酵产物高、中和低各组肿瘤细胞大量坏死,并且高剂量和低剂量组血清中白细胞介素-2和干扰素-r含量显著增加,血管内皮生长因子含量降低,与抑制肿瘤效果具有一定相关性;此外,红缘拟层孔菌固体发酵产物各组可以降低丙二醛含量,提高超氧化物歧化酶、过氧化氢酶、谷胱甘肽过氧化物含量;综上所述,红缘拟层孔菌固体发酵产物具有显著的抗肿瘤和抗氧化作用。     

红缘拟层孔菌发酵菌丝体对小鼠免疫调节的研究

红缘拟层孔菌是具有良好抗肿瘤活性的药用真菌,其抗肿瘤的主要有效成分为3-乙酰氧基-8,24-羊毛甾二烯-21-酸。以红缘拟层孔菌发酵菌丝体及3-乙酰氧基-8,24-羊毛甾二烯-21-酸单体为实验材料,研究了两者对小鼠免疫功能的调节作用,然后测试免疫功能的4个指标来判断红缘拟层孔菌发酵菌丝体和3-乙酰氧基-8,24-羊毛甾二烯-21-酸单体在免疫系统中的影响。结果表明,与对照组比较,红缘层孔菌组的吞噬率、血清溶血素水平、淋巴细胞转化率和吞噬指数均有显著提高;在小鼠淋巴细胞转化实验中,3-乙酰氧基-8,24-羊毛甾二烯-21-酸在低浓度时对小鼠淋巴细胞转化具有良好的促进作用,但随浓度的增加,促进作用逐渐减弱(P0.05)。因此,红缘层孔菌发酵菌丝体具有良好的增强小鼠免疫功能的作用。     

药用拟层孔茵研究进展

对药用拟层孔菌的化学成分、药理活性、培养基优化、HPLC -指纹图谱鉴定以及生物学特性等方面进行了综述,并对药用拟层孔菌的深入研究进行了展望.     

药用拟层孔茵研究进展

对药用拟层孔菌的化学成分、药理活性、培养基优化、HPLC -指纹图谱鉴定以及生物学特性等方面进行了综述,并对药用拟层孔菌的深入研究进行了展望.     

药用拟层孔菌石油醚提取物的GC-MS分析及各相提取物生物活性

通过形态学和分子生物学分析,实验菌株为药用拟层孔菌。采用不同有机溶剂提取药用拟层孔菌子实体,分别获得石油醚、氯仿、乙酸乙酯和甲醇提取物。采用GC-MS方法,分析药用拟层孔菌石油醚提取物中的化学成分,同时对不同有机溶剂提取物的生物活性进行检测。结果表明:药用拟层孔菌石油醚提取物中共鉴定出46个化合物。体外抑制肿瘤细胞活性实验表明,在100μg/m L时,不同有机溶剂提取物都表现出抑制肿瘤细胞活性。其中,石油醚提取物在50μg/m L时,其对NCI-H460和SGC-7901肿瘤细胞的抑制率分别为99.03%和82.57%。甲醇提取物在8mg/m L时,DPPH自由基清除率达93.35%。     

火木层孔菌发酵菌粉及其组分对小鼠移植性肝癌H22的体内生长作用

采用肝癌H22荷瘤小鼠的肿瘤模型,对火木层孔菌(桑黄)Phellinus igniarius发酵菌粉及其各提取物组分的体内抗肿瘤活性进行评价。结果表明,火木层孔菌发酵菌粉及其各提取物组分对荷瘤小鼠肝癌H22都具有一定的抗肿瘤作用并能延长荷瘤小鼠生存期,其中,火木层孔菌发酵菌粉(1,000mg/kg/d)及其组分I(I多糖组分)(360mg/kg/d)具有较为显著的抗肿瘤作用,抑瘤率分别为33.5%和40.3%。组织病理学研究结果表明在菌粉及其多糖组分作用后,肿瘤细胞坏死细胞明显增多,免疫组化检测表明火木层孔菌发酵菌粉及其多糖组分能明显的降低瘤组织中Bcl-2基因蛋白的表达,提高小鼠瘤组织中的Bax基因蛋白的表达。火木层孔菌发酵菌粉及其多糖组分具有较好的体内抗肿瘤活性。     

血红密孔菌高产漆酶菌株的筛选及其对烟梗的生物降解

白腐菌是目前已知的唯一能将木质素彻底降解的微生物,而漆酶在木质素分解过程中起着重要的作用,被广泛应用于农作物秸秆或甘蔗渣等多种类型生物质的生物预处理和生物降解。本研究利用白腐菌产漆酶发酵培养基对30株血红密孔菌Pycnoporus sanguineus菌株进行筛选,得到了多株漆酶高产菌株,并研究了血红密孔菌发酵粗酶液和菌丝对烟梗的生物降解条件。研究结果表明:血红密孔菌及其产生的漆酶表现出了对烟梗木质素较强的生物降解能力。在漆酶浓度为40U/mL、温度30℃、pH4.5的条件下处理24h,烟梗中木质素的降解率可达到50.4%,纤维素和半纤维素的降解率分别为17.5%和17.3%;漆酶浓度为5U/mL、温度30℃、pH4.5的条件下处理48h,木质素降解率可达到65.1%。血红密孔菌菌丝也表现出对烟梗较好的生物降解效果,接种培养7d后烟梗中木质素降解率可达30%以上,21d后木质素的降解率可达79.1%,而纤维素和半纤维素的降解率仅为20%和12%左右。本研究不但为生物质材料的生物预处理和生物降解提供了优质的白腐菌及漆酶资源,还为通过烟梗的生物预处理提高烟草梗丝和卷烟品质提供了重要参数,具有一定的应用前景。     

多孔菌三个中国新记录种

报道了多孔菌3个中国新记录种,其中包括1个新组合种：胭脂红栓孔菌。褐白革孔菌Coriolopsis brunneoleuca采自广西、海南和云南省,该种的主要特征为子实体盖形,菌盖表面浅黄褐色至黄褐色,被绒毛,骨架菌丝和缠绕菌丝有拟糊精反应;婆罗洲榆孔菌Elmerina borneensis采自福建和海南省,子实体平伏,子实层中较多大的菌丝钉,担子纵分隔;胭脂红栓孔菌Trametes coccinea采自吉林省,子实体橘黄色、浅红褐色、红色至砖红色,孔口小。文中对这3个种进行了详细的描述和显微结构绘图并且提供了红色栓孔菌种类（即红孔菌属种类）的检索表。     

血红铆钉菇化学成分和药理活性研究概述

从化学成分、药理活性方面对血红铆钉菇的研究现状进行了综述,其化学成分主要包括多糖类、脂肪酸类、内酯类、香豆素类、酚类、甾类、羟基蒽醌类和萜类等;在药理活性方面,对其抗肿瘤、神经元保护、抗氧化、抗病原菌、促生长以及抗运动病等作用进行了较全面的概述,旨在为今后的研究和开发利用提供基础。     

Exopolysaccharide production and mycelial growth in an air-lift bioreactor using Fomitopsis pinicola

For effective exopolysaccharide production and mycelial growth by a liquid culture of Fomitopsis pinicola in an air-lift bioreactor, the culture temperature, pH, carbon source, nitrogen source, and mineral source were initially investigated in a flask. The optimal temperature and pH for mycelial growth and exopolysaccharide production were 25degrees C and 6.0, respectively. Among the various carbon sources tested, glucose was found to be the most suitable carbon source. In particular, the maximum mycelial growth and exopolysaccharide production were achieved in 4% glucose. The best nitrogen sources were yeast extract and malt extract. The optimal concentrations of yeast extract and malt extract were 0.5 and 0.1%, respectively. K2HPO4 and MgSO4 x 7H2O were found to be the best mineral sources for mycelial growth and exopolysaccharide production. In order to investigate the effect of aeration on mycelial growth and exopolysaccharide production in an air-lift bioreactor, various aerations were tested for 8 days. The maximum mycelial growth and exopolysaccharide production were 7.9 g/l and 2.6 g/l, respectively, at 1.5 vvm of aeration. In addition, a batch culture in an air-lift bioreactor was carried out for 11 days under the optimal conditions. The maximum mycelial growth was 10.4 g/l, which was approximately 1.7-fold higher than that of basal medium. The exopolysaccharide production was increased with increased culture time. The maximum concentration of exopolysaccharide was 4.4 g/l, which was about 3.3-fold higher than that of basal medium. These results indicate that exopolysaccharide production increased in parallel with the growth of mycelium, and also show that product formation is associated with mycelial growth. The developed model in an air-lift bioreactor showed good agreement with experimental data and simulated results on mycelial growth and exopolysaccharide production in the culture of F pinicola.     

Purification and characterization of a thermostable cellobiohydrolase from Fomitopsis pinicola

A screening for cellobiohydrolase (CBH) activity was performed and Fomitopsis pinicola KMJ812 was selected for further characterization as it produced a high level of CBH activity. An extracellular CBH was purified to homogeneity by sequential chromatography of F. pinicola culture supernatants. The molecular mass of the F. pinicola CBH was determined to be 64 kDa by SDS-PAGE and by size-exclusion chromatography, indicating that the enzyme is a monomer. The F. pinicola CBH showed a t1/2 value of 42 h at 70 degrees C and catalytic efficiency of 15.8 mM-1 S-1 (kcat/ Km) for p-nitrophenyl-beta-D-cellobioside, one of the highest levels seen for CBH-producing microorganisms. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase family 7. Although CBHs have been purified and characterized from other sources, the F. pinicola CBH is distinguished from other CBHs by its high catalytic efficiency and thermostability.     

松蜕盾蚧等四种蜕盾蚧分类地位的研究（同翅目：盾蚧科）

将松蜕盾蚧Florinia pinicola Maskell接种于盆栽马尾松上饲养,得到大量标本,通过对这些标本的观察研究,结果发现：臀背边缘管腺的大小．数量以及触角的形态等特征变化很大;研究表明松蜕盾蚧F.pinicola、日本蜕盾蚧F.japonica、柏蜕盾蜕F.externa和霜蜕盾蚧F.prulnosa,实际上是同一个种,即松蜕盾蚧F.pinicola。     

Thiamine increases β-glucosidase production in the newly isolated strain of Fomitopsis pinicola

Aims:  To isolate a high β-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain.
Methods and Results:  A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l⁻¹) improved BGL production in F. pinicola cultures by 3·7-fold to give a specific activity of 114·4 μmol min⁻¹ mg⁻¹ protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported.
Conclusion:  Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812.
Significance and Impact of the Study:  Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.     

Purification and characterization of a thermostable xylanase from Fomitopsis pinicola

An extracellular xylanase was purified to homogeneity by sequential chromatography of Fomitopsis pinicola culture supernatants on a DEAE-sepharose column, a gel filtration column, and then on a MonoQ column with fast protein liquid chromatography. The relative molecular weight of F. pinicola xylanase was determined to be 58 kDa by sodium dodecylsulfate polyacrylamide gel electrophoresis and by size exclusion chromatography, indicating that the enzyme is a monomer. The hydrolytic activity of the xylanase had a pH optimum of 4.5 and a temperature optimum of 70 degreesC. The enzyme showed t(1/2) value of 33 h at 70 degrees C and catalytic efficiency (k(cat) = 77.4 s?1, k(cat)/K(m) = 22.7 mg/ml/s) for oatspelt xylan. Its internal amino acid sequences showed a significant homology with hydrolases from glycoside hydrolase (GH) family 10, indicating that the F. pinicola xylanase is a member of GH family 10.     

Molecular cloning of functional genes for high growth-temperature and salt tolerance of the basidiomycete Fomitopsis pinicola isolated in a mangrove forest in Micronesia

Several functional genes encoding putative proteins, heat shock protein 70, sphingosine phosphate lyase, and Na+/H+ antiporter, were cloned from the basidiomycete Fomitopsis pinicola, a wood-rotting fungus isolated in the tropical mangrove forest of Pohnpei Island of the Federated States of Micronesia. The deduced amino acid sequences of the obtained genes involved in heat shock resistance, lipid synthesis, and salt tolerance showed diverse similarities to other homologous proteins. Molecular phylogenetic trees of these proteins suggested that encoded proteins of the cloned genes of F. pinicola differed remarkably from other homologs in various organisms, even fungal proteins. Putative candidates for other genes related to several cellular metabolisms were also amplified, implying the possible existence of those genes in F. pinicola. This is the first report of possibly functional genes derived from a basidiomycetous mushroom growing in tropical islands such as Micronesia. The genes found in this study might play important roles in the cellular survival of the basidiomycete F. pinicola under severe environmental conditions.     

Spore deposition of wood-decaying fungi: importance of landscape composition

The abundance of many species of wood-decaying fungi has decreased dramatically in Swedish boreal forests over the last century. Therefore, we have investigated the relationship between the spore dispersal of wood-decaying fungi and two key features of landscape composition, namely the amount and age of old forest stands at different spatial scales. Spore deposition was monitored in two regions, using sampling methods based on recording the dikaryotisation of monokaryotic mycelia on nutrient agar and wood discs. The studied species, all mainly confined to Norway spruce in the boreal forest zone, were Fomitopsis pinicola, Fomitopsis rosea , Gloeoporus taxicola , Phlebia centrifuga and Trichaptum laricinum . The study of forestry intensity showed for both regions, that the spore deposition for F. pinicola , F. rosea and G. taxicola was higher in circular plots (radius 2-km) with a high proportion of old Norway spruce forest (>80 yr) than in plots with a lower proportion of old forest. Analysis of the variation in spore deposition of F. rosea and P. centrifuga in relation to the proportion of old spruce forest within 1, 2 and 3 km of the spore sampling point showed that the proportion of old forest within a 3-km radius explained more of the variation than the proportions within 1- and 2-km radii. In addition, the proportion of forest older than 140 yr explained more of the variation than the proportion of younger forests. Thus, the results show that the spore deposition of the studied species strongly depends on the landscape composition at both regional and local scales. Further, the spore deposition at the local scale was best explained by the proportion of >140 yr old spruce forest, which exceeds the common harvest rotation period.     

Constituents of various wood-rotting basidiomycetes

Phytochemical investigation of n-hexane and methanol extracts of fruiting bodies of the wood-rotting fungi Fomitopsis pinicola. Ganoderma lipsiense, Fomes fomentarius and Gloeophyllum odoratum led to the isolation and identification of several triterpene derivatives and some aromatic compounds derived from lignin. These are the new natural products, namely, pinicolic acid E (16alpha-hydroxy-3-oxolanosta-8,24-dien-21-oic acid) and pinicolol C (3-oxolanosta-7,9(11),24-trien-15alpha,21-diol) from the crust of F. pinicola, ganoderenic acid D [(E)-7beta-hydroxy-3,11,15,23-tetraoxolanosta-8,20(22)-di en-26-oic acid] and ganoderic acid N (7beta,20-dihydroxy-3,11,15,23-tetraoxolanost-8-en-26-oic acid) from G. lipsiense and ergosterol peroxide (5alpha,8alpha-epi-dioxyergost-6-en-3beta-ol) as well as ergost-7-en-3-one from F. fomentarius. From G. odoratum, dehydroeburicoic acid [24-methylene-3-oxolanosta-7,9(11)-dien-21-oic acid], the dimethylacetal of 4,4,14alpha-trimethyl-24-oxo-5alpha-chol-8-en-21-oic acid and some aromatic compounds, of which 1-(4'-methoxyphenyl)-1,2-ethandiol is a new natural product, were isolated. Furthermore, a complete set of 13C NMR data of the steryl esters 3beta-linoleyloxyergosta-7,24(28)-diene, 3beta-linoleyloxyergosta-7,24-diene and 3beta-linoleyloxyergost-7-ene, which could be identified as a mixture in all investigated fungi, could be recorded. It was proved by HPLC and TLC investigations, that the crust on top of the fruiting bodies of F. pinicola consists of lanostane derivatives.     

Review of the Nearctic genus Lacconotus LeConte (Coleoptera, Mycteridae, Eurypinae)

Lacconotus LeConte, the sole Nearctic representative of the eurypine Mycteridae, is revised, based on morphological features of adults. The following syn. n. is proposed: Lacconotus pallidus Van Dyke, 1928 = Lacconotus pinicola Horn, 1879. The former is a light-colored form with a southern California distribution. A subgen. n.,Alcconotus, is described for Lacconotus pinicola, producing the following comb. n.: Lacconotus (Alcconotus) pinicola (Horn). A lectotype is designated for Lacconotus pinicola. A key separating the two subgenera and species is provided, as are photographs and illustrations of salient structures of adults, and maps showing collection localities. Lacconotus punctatus is newly recorded in Alabama, Arkansas, Massachusetts, Oklahoma, Texas, and Wisconsin; Lacconotus pinicola is newly recorded in Arizona and Utah in the USA, and Baja California Norte in Mexico. Phenology information shows a north-to-south gradation in occurrence time.     

Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.