灵芝蛛网病病原菌及其生物学特性

为明确吉林省蛟河市灵芝Ganoderma lingzhi栽培主产区发生的疑似灵芝蛛网病的病原菌,作者通过罹病灵芝子实体病原物的分离纯化、致病性测定、形态学和分子生物学鉴定以及病原菌的生物学特性研究,证明引起吉林省蛟河市灵芝蛛网病的病原菌为嗜菌枝葡霉Cladobotryum mycophilum。该菌营养体最适生长条件为温度25℃、pH 5、蔗糖作碳源、酵母浸粉作氮源,光照对菌丝体生长有一定的抑制作用,完全黑暗最适宜生长。本文研究结果为进一步研究该病害的发生规律和防治措施提供了理论参考。     

Analysis of the Candida albicans proteome. I. Strategies and applications

The alarming incidence of invasive candidiasis, predominantly among the recent expanding immunocompromised population, the appearance of antifungal-drug resistance, and the lack of specific diagnostic tests for it have demanded more impactful research into Candida albicans pathogenicity. Proteomic approaches can provide accurate clues about its biological complexity. Indeed, initial C. albicans proteome analyses have focused on the understanding of dimorphism, host responses, the cell wall, virulence factors and drug resistance, among others. This review aims to briefly outline the technology available for proteomics-based studies, surveying the main proteomic approaches applied to C. albicans research. Prefractionation techniques, two-dimensional gel electrophoresis and mass spectrometry continue to be the backbone of proteomic projects. Emerging strategies for protein separation, quantification and identification may, however, challenge the pivotal position of 2D-PAGE. Regardless of this, since we are now approaching the completion and annotation of C. albicans genome sequencing, systematic characterization of the proteome of this fungal pathogen, although still in its early stages, heralds an exciting expansion of our knowledge in years to come.     

The effect of selenium enrichment on baker's yeast proteome

The use of regular yeast (RY) and selenium-enriched yeast (SEY) as dietary supplement is of interest because the Nutritional Prevention of Cancer (NPC) trial revealed that SEY but not RY decreased the incidence of prostate cancer (PC). Using two-dimensional difference in gel electrophoresis (2D-DIGE)-tandem mass spectrometry (MS/MS) approach, we performed proteomic analysis of RY and SEY to identify proteins that are differentially expressed as a result of selenium enrichment. 2D-DIGE revealed 96 candidate protein spots that were differentially expressed (p ≤ 0.05) between SEY and RY. The 96 spots were selected, sequenced by LC/MS/MS and 37 proteins were unequivocally identified. The 37 identified proteins were verified with ProteinProphet software and mapped to existing Gene Ontology categories. Furthermore, the expression profile of 5 of the proteins with validated or putative roles in the carcinogenesis process, and for which antibodies against human forms of the proteins are available commercially was verified by western analysis. This study provides evidence for the first time that SEY contains higher levels of Pyruvate Kinase, HSP70, and Elongation factor 2 and lower levels of Eukaryotic Translation Initiation Factor 5A-2 and Triosephosphate Isomerase than those found in RY.     

Effect of five fungicides with different modes of action on cobweb disease (Cladobotryum mycophilum) and mushroom yield

The fungicides chlorothalonil, metrafenone, prochloraz‐Mn, thiabendazole and thiophanate‐methyl were tested in vitro and in vivo for their effect on Cladobotryum mycophilum, the mycoparasite that causes cobweb disease in white button mushroom. In vitro experiments showed that metrafenone (EC₅₀= 0.025 mg L^?1) and prochloraz‐Mn (EC₅₀= 0.045 mg L^?1) were the most effective fungicides for inhibiting the mycelial growth of C. mycophilum. Selectivity indexes of the tested fungicides on both C. mycophilum and Agaricus bisporus indicated that metrafenone was also the most selective fungicide, while chlorothalonil was the most toxic fungicide against A. bisporus mycelium. The in vivo efficacy of fungicides for controlling cobweb was evaluated in three mushroom cropping trials, which were artificially inoculated with C. mycophilum (10⁶ conidia m^?2). Prochloraz‐Mn provided good control, although the surface colonised by cobweb reached 12% by the end of the crop cycles. None of the inoculated cropping trials treated with metrafenone showed any cobweb disease symptoms, and neither were any significant phytotoxic effects on mushroom yield recorded. These results indicated that metrafenone can be used as an alternative to prochloraz‐Mn in the control of cobweb disease.     

Incidence,identification and pathogenicity of Cladobotryum mycophilum,causal agent of cobweb disease on Agaricus bisporus mushroom crops in Spain

Between 2008 and 2011, outbreaks of cobweb were observed in commercial white button mushroom (Agaricus bisporus) crops in Castilla‐La Mancha (Spain). In the last 3 years, the presence of the disease has notably increased resulting in serious economic losses. Based on morphological and genetic analyses, the casual agent of cobweb was identified as Cladobotryum mycophilum. A. bisporus mushroom crops were surveyed over a 2‐year period to estimate the incidence of cobweb. The presence of the disease was detected in 32% of the mushroom crops observed, being of particular concern in autumn (44% of crops infected) and winter (37%). As regards the casing material, the percentage of crops affected by cobweb was 34% in crops using mineral casing and 29% in those cased with a peat‐based casing, with no statistical relationship between the casing and the presence of cobweb. Two cropping trials inoculated with C. mycophilum were set up to evaluate the pathogenicity of the causal agent of cobweb in three peat‐based casings (C1, C2 and C3). The effect of cobweb on mushroom productivity was evaluated by comparing mushroom production and the cobweb patches detected in the casing soil. The decrease in total yield of mushrooms attributed to cobweb reached 12.9% with C2, and the crop area colonised by cobweb reached a final percentage of 36% with C3.     

乳杆菌对数生长期培养基滤液核酸组分分析

目的通过对乳杆菌对数生长期培养基滤液中核酸组分的分析,阐明乳杆菌DM9811对数生长期培养基滤液中核酸的性质。方法应用核酸的分离、纯化及电泳分析技术。结果乳杆菌对数生长期培养基滤液中核酸组分为RNA,其片段大小为100 bp左右。乳杆菌对数生长期培养基滤液中核酸组分为RNA,为对数期产生且呈时间依赖关系。结论核酸组分不仅仅是遗传信息的载体,还可能作为有效的信息分子。     

纳豆枯草杆菌培养滤液对家兔离体肠平滑肌的作用

目的观察纳豆枯草杆菌培养滤液（culture filtrate,CF）对家兔离体肠平滑肌的作用,并初步探讨其作用机制。方法制备兔离体回肠标本,分别记录肠平滑肌的正常收缩张力和收缩频率作为给药前对照,然后按累积剂量分别加入CF小剂量（每次0．2mL）、CF大剂量（每次0．5mL）、肉汤（每次0．5mL）,给药间隔3min,共给药8次,并描记收缩曲线。观察不同剂量cF对肠平滑肌的作用。另取肠段按毛果芸香碱、CF或阿托品、再毛果芸香碱的顺序给药,观察CF对M胆碱受体的作用。结果CF小剂量组在累积给药达1．6mL时,CF大剂量组在累积给药达3．5mL和4．0mL时,兔离体肠平滑肌收缩张力下降,与给药前比较差异有统计学意义（P〈0．05）,其余各点差异均无统计学意义（P〉0．05）。CF小剂量组在累积给药达1．2、1．4、1．6mL时,CF大剂量组在累积给药达4．0mL时,兔离体肠平滑肌收缩频率明显降低,与给药前比较差异有统计学意义（P〈0．05或P〈0．01）,其余各点差异均无统计学意义（P〉0．05）。CF或阿托品可明显对抗毛果芸香碱引起的兔离体肠平滑肌收缩张力的增加（P〈0．05或P〈0．01）,同时CF还能使其收缩频率明显减少（P〈0．01）。结论纳豆枯草杆菌CF能明显抑制兔离体肠平滑肌蠕动,其作用机制可能与阻断M胆碱受体有关。     

Correlation of probiotic Lactobacillus salivarius growth phase with its cell wall-associated proteome

Lactobacillus salivarius subsp. salivarius UCC118 is a probiotic bacterium that was originally isolated from human intestinal tissues and was subsequently shown in a pilot study to alleviate symptoms associated with mild-moderate Crohn's disease. Strain UCC118 can adhere to animal and human intestinal tissue, and to both healthy and inflamed ulcerative colitis mucosa, irrespective of location in the gut. In this study, an enzymatic technique has been combined with proteomic analysis to correlate bacterial growth phase with the presence of factors present in the cell wall of the bacterium. Using PAGE electrophoresis, it was determined that progression from lag to log to stationary growth phases in vitro correlated with increasing prominence of an 84kD protein associated with in vitro adherence ability. Isolated proteins from the 84kD band region were further separated by two-dimensional electrophoresis, resolving this band into 20 individual protein spots at differing isoelectric points. The protein moieties were excised, trypsin digested and subjected to tandem mass spectrometry. The observed proteins are analogous to those reported to be associated with the Listeria monocytogenes cell-wall proteome, and include DnaK, Ef-Ts and pyruvate kinase. These data suggest that at least some of the beneficial attributes of probiotic lactobacilli, and in particular this strain, may be due to nonpathogenic mimicry of pathogens and potentially be mediated through a form of attenuated virulence.     

Antimicrobials and in vitro systems: antibiotics and antimycotics alter the proteome of MCF-7 cells in culture

Cell culture is widely used to study gene or protein changes in response to experimental conditions. The value of such experiments depends on stringent control and understanding of the in vitro environment. Despite well-documented evidence describing toxic effects in the clinical setting, antibiotics and antimycotics are routinely used in cell culture without regard for their potential toxicity. We cultured MCF-7 breast cancer cells in the presence/absence of antibiotics (penicillin/streptomycin) and/or the antimycotic amphotericin B. Differential protein expression was assessed using 2D-DIGE and MALDI-MS/MS. Antibiotics caused 8/488 spots (1.3% of the protein) to be generally down-regulated. The affected proteins were principally chaperones and cytoskeletal. In marked contrast, amphotericin B induced a more dramatic response, with 33/488 spots (9.5% of the total protein) generally up-regulated. The proteins were mostly involved in chaperoning and protein turnover. Combining antibiotics and amphotericin B had little overall effect, with only one (unidentified) protein being up-regulated. As this study identifies differential protein expression attributable to antibiotics/antimycotics, we urge caution when comparing and interpreting proteomic results from different laboratories where antibiotics/antimycotics have been used. We conclude that as antibiotics and antimycotics alter the proteome of cultured cells in markedly different ways their use should be avoided where possible.     

Harry Belafonte and the secret proteome of coconut milk

The proteome of coconut milk has been extensively mapped via capture at three pH values with combinatorial peptide ligand libraries (CPLL). A grand total of 307 unique gene products could be listed, 200 discovered via CPLL capture, 137 detected in the control, untreated material and 30 species in common between the two sets of data. This is by far the most extensive mapping of coconut milk, in which, up to the present, only a dozen proteins were known, those belonging to the high- to very-high abundance class. The database of coconut contains only 106 proteins: of those, only six are listed in our table. The vast majority of the classified proteins, thus, has been identified only by homologies with sequences deposited in the general viridiplantae database. This unique set of data could be the starting point for nutritionists and researchers involved in nutraceutics for enucleating some proteins responsible for some of the unique beneficial health effects attributed to coconut milk.     

Analysis of the Candida albicans proteome. II. Protein information technology on the Net (update 2002)

Candida albicans is an important fungal model organism of noteworthy clinical interest in modern medicine. Different initiatives addressing its sequencing and physical mapping have been carried out. The C. albicans genome sequence is currently near to completion at Stanford University, heralding new challenges in proteomic research and functional analyses of its gene products. This review presents an update of the most relevant data resources that are available through the World Wide Web to scientists working in the area of the analysis of the C. albicans proteome. An overview of the current status of the main universal protein sequence databases and specialized data collections for C. albicans is given. Various issues of the single public C. albicans 2D-PAGE database are also described, highlighting the significance of setting up graphical query interface-based databanks to visualize 2D-PAGE images through the Net. Finally, we also emphasize the pressing need to create a "cyber-bioknowledge library" that will integrate all the databases developed at the different levels for the understanding of life processes as well as bioinformatic tools for interpreting this deluge of data generated through the Internet.     

Azoreductase and dye detoxification activities of Bacillus velezensis strain AB

Azo dyes are known to be a very important and widely used class of toxic and carcinogenic compounds. Although lot of research has been carried out for their removal from industrial effluents, very little attention is given to changes in their toxicity and mutagenicity during the treatment processes. Present investigation describes isolation of a Bacillus velezensis culture capable of degrading azo dye Direct Red 28 (DR28). Azoreductase enzyme was isolated from it, and its molecular weight was found to be 60 kDa. The enzyme required NADH as cofactor and was oxygen-insensitive. Toxicity and mutagenicity of the dye during biodegradation was monitored by using a battery of carefully selected in vitro tests. The culture was found to degrade DR28 to benzidine and 4-aminobiphenyl, both of which are potent mutagens. However, on longer incubation, both the compounds were degraded further, resulting in reduction in toxicity and mutagenicity of the dye. Thus, the culture seems to be a suitable candidate for further study for both decolourization and detoxification of azo dyes, resulting in their safe disposal. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Comparative proteome approach demonstrates that platelet-derived growth factor C and D efficiently induce proliferation while maintaining multipotency of hMSCs

This is the first study that comprehensively describes the effects of the platelet-derived growth factor (PDGF) isoforms C and D during in vitro expansion of human mesenchymal stem cells (hMSCs). Our results show that PDGFs can enhance proliferation of hMSCs without affecting their multipotency. It is of great value to culture and expand hMSCs in a safe and effective manner without losing their multipotency for manipulation and further development of cell-based therapies. Moreover, differential effects of PDGF isoforms have been observed on lineage-specific differentiation induced by BMP2 and Vitamin D3. Based on label-free LC-based quantitative proteomics approach we have furthermore identified specific pathways induced by PDGFs during the proliferation process, showing the importance of bioinformatics tools to study cell function.     

Isolation and identification of secondary metabolites from hexane extract of culture filtrate of Bacillus licheniformis MTCC 7445

Hexane extract of cell free culture filtrate of Bacillus licheniformis MTCC 7445 isolated from rhizoshere soil of a resistant tomato plant showed antifungal activity against a number of soilborne and human pathogenic fungi. Maximum activity was observed against Botrytis cinerea (ED₅₀ = 23.79 μg/ml), Candida albicans (ED50 33.45 μg/ml) and Microsporum canis (ED₅₀ = 39.02 μg/ml). Metabolites such as 1-methyl pyrrolidene, 1-methyl cyclohexene, 4,4-dimethyl cyclohexane, ethyl-4-ethoxybenzoate, 2-butoxyethanol, naphthalene, ter butyl benzene and phenoxy acetic acid were identified by GC-MS and comparing the mass spectrum with the NIST library.     

Proteome characterization of the culture supernatant of Mycobacterium bovis in different growth stages

This study aimed to identify proteins secreted by Mycobacterium bovis into culture medium at different stages of bacterial growth. A field strain of M. bovis was grown in Middlebrook 7H9 media and culture supernatant was collected at three-time points representing three different phases of growth (early exponential, late exponential, and stationary phases). Supernatants were double filtered, digested by trypsin and analyzed by LC-MS/MS. The study found 15, 21, and 16 proteins in early, mid and late growth phases, respectively. In total, 22 proteins were identified, 18 of which were reported or predicted to have a cell wall or extracellular localization. To our knowledge, this is the first study to identify proteins secreted into the culture medium by a field strain of M. bovis in three different stages of growth. The dataset generated here provides candidate proteins with the potential for the development of serological diagnostic reagents or vaccine for bovine tuberculosis. Data are available via ProteomeXchange with identifier PXD017817.