Alternaria alternata NADP-dependent mannitol dehydrogenase is an important fungal allergen.

BACKGROUND: Alternaria alternata is one of the most important allergenic fungi worldwide. Mannitol dehydrogenase (MtDH) has previously been shown to be a major allergen of Cladosporium herbarum and cross-reactivity has been demonstrated for several fungal allergens. OBJECTIVE: The present study's objective was to clone the MtDH from an A. alternata cDNA library, express and purify the recombinant non-fusion protein and test its IgE-binding properties. Methods A cDNA library prepared from A. alternata hyphae and spores was screened for mannitol dehydrogenase by DNA hybridization with the radioactively labelled C. herbarum homologue as a probe. The resulting clone was sequenced and heterologously expressed in Escherichia coli as a recombinant non-fusion protein, which was purified to homogeneity and analysed for its IgE-binding capacity. RESULTS: The coding sequence of the full-length cDNA clone comprises 798 bp encoding a protein with a molecular mass of 28.6 kDa and a predicted pI of 5.88. Protein sequence analysis revealed an identity of 75% and a homology of 86% between the MtDHs of A. alternata and C. herbarum. The functional mannitol dehydrogenase was expressed in the E. coli strain BL21(DE3) transformed with the vector pMW172 and purified to homogeneity. The enzyme catalyses the NADPH-dependent conversion of d-fructose to d-mannitol. In IgE-ELISA and immunoblots, MtDH is recognized by 41% of A. alternata-allergic patients. In vivo immunoreactivity of the recombinant MtDH was verified by skin prick testing. Finally, inhibition-ELISA experiments confirmed cross-reactivity between the MtDHs of A. alternata and C. herbarum. CONCLUSION: Mannitol dehydrogenase (Alt a 8) represents an important new allergen of the ascomycete A. alternata that might be suitable for improving diagnostic and therapeutic procedures.     

交链孢酚诱发人胚食管上皮癌的研究

将体外培养的水囊引产的人胎儿食管用交链孢酚(Alternariol,AOH)处理24h,再培养两周,分别接种于裸小鼠皮下和腹腔内。皮下接种的3个食管组织块中之一形成鳞状细胞癌,其余2个和接种于腹腔的2个食管组织块上皮细胞呈明显增生或乳头状增生。空白对照组的食管组织块未见增生。溶剂对照组者仅呈轻度增生。结果提示互隔交链孢霉在人食管癌病因学中起着重要作用。     

交链孢酚激活人胎食管上皮组织细胞癌基因的研究

本文报道食管癌高发区粮食中优势污染菌互隔交链孢霉代谢产物交链孢酚(AOH)对人胎儿食管上皮组织癌基因的激活。结果表明,体外培养的人胎儿食管上皮组织经10gg/ml AOH作用4h后,其DNA具有转化活性,可转化NIH/3T3细胞。一轮转化率为0.17Foci/μg DNA,二轮转化率为0.58Foci/μg DNA(P<0.01)。转化细胞中含有人特异性高重复序列Alu,提示转化系人DNA转染所致。Southern blot检测表明,AOH处理后的人胎儿食管上皮组织内有H-ras和myc基因的扩增,经其转染的NIH/3T3细胞中亦有H-ras基因持久而稳定的扩增。对照组胎儿食管上皮和正常NIH/3T3细胞中均未发现相应的同源序列,说明正常胎儿食管组织中的H-ras和myc基因可经AOH短期处理而激活。该结果对AOH可能系人食管癌病因之一提供了直接证据。     

互隔交链孢霉提取物对淋巴细胞核仁形成区及细胞增殖周期的影响

本实验应用人类核仁形成区(NORs)选择性银染方法和细胞光度术,观察了互隔交链孢霉两菌株提取物261-B_2-3和C_(12)b_3-2对人淋巴细胞NORs活性的影响以及后者对细胞增殖周期移行的影响。结果显示,应用不同浓度的261-B_2-3和C_(12)b_3-2提取物处理细胞后,细胞携带Ag-NORs的染色体数明显减少,与对照组比较差异极显著(P<0.01)。表明两株提取物能明显抑制淋巴细胞NORs的活性。细胞暴露于不同浓度的C_(12)b_3-2提取物后,G_1%明显增多,S%和G_2+M%显著减少,与对照相比差异极显著(P<0.01)。说明C_(12)b_3-2提取物可能作用于G_1后期或S早期,延缓和阻滞了细胞周期的移行。     

Characterization of lipopeptide produced by Bacillus altitudinis Q7 and inhibitory effect on Alternaria alternata

This study identified the antifungal metabolites produced by Bacillus altitudinis Q7 against Alternaria alternata and investigated the antifungal activity and antifungal action. Lipopeptide, the important secondary metabolites were identified by Fourier transform infrared (FTIR) and liquid chromatography-mass spectrometry as lichenysin. The antifungal activity of lipopeptide on A. alternata was determined by microdilution technique, and its minimum inhibitory concentration was 1.2 mg/ml. Stability test showed that lipopeptide had excellent temperature and pH resistance. To investigate whether lichenysin acted on the cell membrane and changed its permeability, the ultra-violet absorption of protein and nucleic acid were measured using a colorimetric method. The antifungal metabolites produced by B. altitudinis Q7 was lichenysin, which showed stable antifungal activity in the extreme environments. Lichenysin could inhibit A. alternata by altering the permeability of cell membrane, leading to the outflow of proteins and nucleic acids from the cytoplasm. This research suggests the lipopeptide from B. altitudinis Q7 is a potential biological control agent against A. alternata.     

Microbial transformation of asiatic acid by Alternaria longipes

Asiatic acid is a major pentacyclic triterpene isolated from Centella asiatica. It shows a variety of bioactivities. In order to obtain its derivatives, potentially useful for detailed pharmacological studies, the substrate was subjected to incubations with selected micro-organisms. In this work, asiatic acid was converted into three new compounds: 2α,3β,23,30-tetrahydroxyurs-12-ene-28-oic acid (1), 2α,3β,22β,23-tetrahydroxyurs-12-ene-28-oic acid (2), and 2α,3β,22β,23,30-pentahydroxyurs-12-ene-28-oic acid (3) by the fungus Alternaria longipes AS 3.2875. The structures of the three metabolites were determined by 1D and 2D NMR spectral data.     

柳珊瑚共附生真菌Alternaria sp.(WZL003)抑菌化合物的分离及其作用机制初步研究D

目的研究1株柳珊瑚共附生真菌具有抑菌活性次级代谢产物及抑菌作用机制。方法采用活性追踪分离的方法,运用硅胶柱层析、Sephadex LH-20凝胶柱层析和半制备HPLC等手段,对采自中国南海的柳珊瑚Echinogorgia rebekka中分离获得的共附生链格孢霉属真菌Alternaria sp.(WZL003)的发酵产物进行分离和纯化,利用NMR、MS等波谱方法,并与文献对照,确定化合物的结构;以海洋病原菌和污损菌鳗弧菌Vibrio anguillarum为对象研究化合物的抑菌作用机制。结果从该菌发酵液的乙酸乙酯提取物中,分离获得1个对V.anguillarum具有较强抑菌活性的蒽醌类化合物macrosporin。结论初步的抑菌作用机制研究发现,该化合物能破坏鳗弧菌的细胞壁和细胞膜,导致细胞通透性增加,胞内大量内容物外泄,最终导致菌体死亡,从而起到抑菌作用。本研究首次报道了macrosporin对鳗弧菌的抑菌作用及其作用机制。     

林县互隔交链孢霉素x_4结构鉴定

从林县粮食中分离的优势菌株互隔交链孢霉培养物中提取出七种代谢物,对其中成份之一x_4,经质谱、红外光谱、核磁共振光谱测定,同时与标准品及人工合成品作比较,证明x_4成份是交链孢酚(Alternariol AOH)。     

Marker antigens to assess exposure to molds and their allergens. II. Alternaria alternata

The estimation of exposure to molds and their products in the indoor environment, which may lead to the occurrence of allergies or respiratory complaints, by means of enumeration of viable parts is inadequate. Therefore, other methods must be developed. When grown under various circumstances (22 degrees C and 30 degrees C, high and low water activity) under laboratory conditions, Alternaria alternata produces one antigen that can be found under all studied growth conditions in extracts of the water-soluble portion of the mycelium. This common antigen may serve as marker antigen for exposure to A. alternata and its allergens. In extracts of the culture filtrate, three antigens, designated index antigens, have been identified that together may have the function of marker for the exposure to allergens of A. alternata.