五种药用石斛内生真菌抑制HIV-1整合酶活性研究

目的 评价5种药用石斛内生真菌发酵产物抑制HIV-1整合酶的活性.方法 将分离自石斛的202株内生真菌提取物共404个采用高通量ELISA法评价其抑制HIV-l整合酶活性;对抑制活性大于100％的样品进行量效关系考察并进行体外抑制肿瘤细胞活性筛选.结果 筛选得到19个对HIV-l整合酶抑制活性大于80％的样品,其中样品5119F、5297F、5097F、5140J和5211F的抑制率分别达到117.96％、113.53％、108.62％、103.74％和109.02％,其对应的IC50值分别为0.02024、0.003125、0.00862、0.01007 和 0.01192 mg/ml.结论石斛属药用植物内生真菌是一个潜在的、丰富的用于筛选HIV-1整合酶抑制剂的资源库,值得进一步研究和开发.     

细叶石斛的位点特异性PCR鉴别(英文)

根据细叶石斛及其它37种枫斗类和黄草类石斛的rDNAITS序列,我们设计了位点特异性PCR鉴别引物XY-JB01S和XY-JB01X,对细叶石斛进行了成功的DNA分子鉴别。在进行位点特异性鉴别PCR之前,首先运用扩增ITS区的通用引物P1、P2对模板DNA进行扩增,以验证模板的可靠性和扩增的合适浓度。当退火温度上升为64℃,只有细叶石斛的模板DNA能被扩增出来,而其它的37种石斛属植物均为阴性。该鉴别反应重复性好,已在鉴别细叶石斛中发挥重要作用。与DNA测序鉴别方法相比,位点特异性PCR具有简单、省时、高效、准确等优点。     

Antifungal activity of endophytic fungi from Cupressaceae against human pathogenic Aspergillus fumigatus and Aspergillus niger

Aspergillus is a fungal genus that strongly affects health of humans, animals, and plants worldwide. Endophytes are now widely considered as a rich source of bioproducts with potential uses in medicine, agriculture, and bioindustry. Cupressaceae plant family hosts a variety of bioactive ascomycetous endophytes. In this study, antifungal activity of a number of such endophytes were investigated against human pathogenic fungi Aspergillus fumigatus and Aspergillus niger. To this end, 16 superior bioactive endophytic fungi from Cupressaceae were used, including Alternaria alternata, Alternaria pellucida, Ascorhizoctonia sp., Aspergillus fumigatus, Aspergillus niger, Aurobasidium sp., Cladosporium porophorum, Fusarium oxysporum, Penicillium viridicatum, Phoma herbarum, Phoma sp., Pyrenochaeta sp., Trichoderma atroviride, Trichoderma atroviride and Trichoderma koningii. In vitro bioassays indicated anti-Asperilli activity of the endophytic fungi in dual cultures. Most notably, Trichoderma koningii CSE₃₂ and Trichoderma atroviride JCE₃₃ showed complete growth inhibition of both A. niger and A. fumigatus, within 3 to 7 days. Also, volatile compouds (VOCs) of T. koningii CSE₃₂ and T. atroviride JCE₃₃ exhibited 33–100% growth inhibition of A. niger, whithin 3 days. Moreover, on the day 7, growth of A. niger was less affected than that of A. fumigatus. In general, it appears that there is a direct relationship between the exposure time and the inhibitory activity of endophytes VOCs on the growth of target Aspergillus species. Furthremore, the extracellular secondary metabolites (SMs) of four selected fungal endophytes exhibited anti-Aspergillus activity at all treatment levels as shown by Agar-diffusion assay. SMs from T. koningii CSE₃₂ and Pyrenochaeta CSE₁₃₄ showed strongest activities against A. niger, and SMs from T. koningii CSE₃₂ and F. oxysporum CAE₁₄ showed strongest activities against A. fumigatus. In conclusion, given the globally recognized issue of antibiotic resistance and the urge to discover new antimicrobial substances, our findings provide new insights into the potential use of Cupressaceae's endophytic fungi in antifungal-based drug discovery programs.     

Inhibition of inflammatory mediators and reactive oxygen and nitrogen species by some depsidones and diaryl ether derivatives isolated from Corynespora cassiicola,an endophytic fungus of Gongronema latifolium leaves

Abstract

In this study, some depsidones and diaryl ether derivatives isolated from Corynespora cassicola, a fungi endophyte of Gongronema latifolium, were assessed for their anti-inflammatory potentials. The isolated metabolites corynesidone A (1), corynesidone C (2), corynesidone D (3) and corynether A (4) were screened for their effects on tumour necrosis factor-α (TNF-α), inducible nitric oxide (iNO), and reactive oxygen species (ROS) and reactive nitrogen species (RNS) production by stimulated RAW264.7 macrophages. Concentration of 1, 2, 3 and 4 up to 100?μM did not remarkably affect the viability of treated macrophages. The compounds were found to cause a concentration-dependent decrease in lipopolysaccharide-induced TNF-α and iNO in RAW264.7 cells. Pre-treatment with 100?μM of 1, 2, 3 and 4 suppressed iNO by as much as 96.28%, 95.71%, 78.14% and 73.28%; with IC₅₀ of 8.16, 9.49, 15.29 and 26.52?μM, respectively. Similarly, pre-treatment with 100?μM of 1, 2, 3 and 4 caused an inhibition of 99.17%, 99.59%, 95.02% and 74.07% in the formation of iNO production, respectively, with IC₅₀ of 1.88, 3.99, 7.48 and 37.22?μM. Treatment of with compounds 1–4 (10, 30 and 100?µM) followed by stimulation with phorbol 12-myristate 13-acetate (1?µM) caused significant (p?<?0.05) suppression of ROS/RNS-evoked chemiluminescence of luminol by as much as 100.96?±?1.88%, 98.59?±?1.38%, 87.35?±?1.41% and 79.22?±?0.30%, respectively at 100?µM. The depsidone derivatives (1–4) showed more potent inhibition of TNF-α and NO production and better scavenging ROS/RNS than the diaryl ether derivative (4). These chemical scaffolds can serve as suitable lead molecules for further development into novel anti-inflammatory and/or anti-cancer agents.     

Keratinophilic fungi from the feathers of Emu (Dromaius novaehollandiae) in Maharastra India

A total of 70 feathers samples of Emu (Dromaius novaehollandiae) were collected from 7 Emu farms situated at two districts (Raigad and Thane) of Maharashtra (India) and screened for resident keratinophilic fungi. Among them, 44 isolates were recovered and identified by evaluating characteristic macro- and micro-morphological features. Further gene products corresponding to the ITS1-5.8S-ITS2 rDNA region from all isolates were amplified and sequenced. Homology search was performed using BLAST program against non-redundant nucleotide database, and significantly matched DNA sequences deposited to the NCBI Gene Bank for reference purposes. Eight identified fungal species belongs to 7 different genera named as Aphanoascus terreus Ac_MW577456 (21.43%), Microsporum gypseum Ac_MW580920 (14.29%), Ctenomyces serratus Ac_MW577459 (10.0%), Uncinocarpus orissi Ac_MW577461 (5.17%), Aphanoascus verrucosus Ac_MW577458 (4.29%), Gymnascella dankaliensis Ac_MW577460 (2.86%), Gymnoascoideus petalosporus Ac_MW577462 (2.86%) and Arthroderma tuberculatum Ac_MW577457 (1.43%).     

Keratinophilic fungi from the vicinity of salt pan soils of Sambhar lake Rajasthan (India)

Forty soil samples were collected from seven sites in the vicinity of Sambhar lake Rajasthan, India and screened for the presence of keratinophilic fungi using hair baiting techniques for isolation. Seventeen isolates were recovered and identified. The cultures were identified by recognition of their macro- and micro- morphological features. Their identification was confirmed by BLAST using ITS1-5.8S-ITS2 rDNA region and sequences have been deposited in NCBI data base. A total of 34 species belonging to 29 genera were isolated. Among the dermatophytes and related keratinophilic fungi Chrysosporium indicum was predominant followed by Ctenomyces serratus, C. tropicum, Keratinophyton durus, Auxarthron conjugatum, Gymnascella dankaliensis, Gymnoascoideus petalosporus and Uncinocarpus reesii. Twenty-six species belonging to 22 genera represented other species. Our study indicates that keratinophilic fungi and species are found in the soils at the vicinity of the Sambhar Lake, and human activities can be the potential source of pathogenic fungi.     

Hepatitis C Virus and its Genotypes in Chronic Liver Disease Patients from Meghalaya,Northeast India

Background and Objectives: Hepatitis C virus (HCV) is an important cause of chronic liver disease (CLD). Although Northeast India is believed to be a HCV hotspot, the proportion of HCV infection and the distribution of HCV genotypes in CLD cases from the region are not known. The objectives of the study were to determine the proportion of HCV infection in newly diagnosed CLD patients from Meghalaya, Northeast India, and further investigate the HCV genotype distribution in those patients. Materials and Methods: The aetiology of CLD was evaluated in 196 newly diagnosed patients, recruited consecutively over a period of 1 year in a medical college hospital from Meghalaya. Those positive for HCV infection were genotyped, and the mode of transmission of the virus was investigated. Results: A considerable proportion (43 patients, 21.9%) of CLD patients were positive for HCV (95% confidence interval [CI]: 16.7%–28.2%). Other leading causes of CLD were alcohol (36.32%) and hepatitis B virus infection (39.3%). Genotype 3 was the most prevalent (48.7%, 95% CI: 33.9%–63.8%), followed by genotype 6 (30.8%, 95% CI: 18.6%–46.6%) and genotype 1 (20.5%, 95% CI: 10.8%–35.5%). The frequency of genotype 6 was remarkably higher than in the other regions of India. Injecting drug use appeared to be the most common mode (28 patients) of acquiring HCV. This was true irrespective of the genotype. Conclusions: The presence of HCV in newly diagnosed CLD cases from Meghalaya was considerable. The genotype distribution of HCV was distinct from the other regions of India.     

In vitro antifungal activity and mechanism of action of chitinase against four plant pathogenic fungi

To determine why chitinase has different antifungal activity on different pathogenic fungi in vitro, we purified recombinant rice chitinase from Pichia pastoris and investigated its antifungal activity against four fungi - Rhizopus stolonifer (Ehrenb. et Fr.) Vuill, Botrytis squamosa Walker, Pythium aphanidermatum (eds.) Fitzp, and Aspergillus niger van Tiegh. Scanning electron microscopy (SEM) and Fourier transform infrared spectroscopy (FTIR) were used to analyze the surface microstructure and proportion of chitin in the cell wall of the four fungi, respectively. The results showed that the chitinase exhibited different antifungal activities against the four fungi, which was directly correlated to the surface microstructure and the proportion of chitin in the fungal cell wall. It will help understanding the antifungal mechanism of the recombinant chitinase and further determining its application scope on crop protection and post-harvest storage of fruits and vegetables. ((c) 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).     

Genetic differentiation and phylogenetic potential of Ty3/Gypsy LTR retrotransposon markers in soil and plant pathogenic fungi

Genetic diversity studies are crucial for understanding the genetic structure and evolutionary dynamics of fungal species and communities. Fungal genomes are often reshaped by their repetitive components such as transposable elements. These elements are key players in genomic rearrangements and are ideal targets for genetic diversity and evolutionary studies. Herein, we used three Ty3/Gypsy long terminal repeat retrotransposons, Grasshopper, Maggy, and Pyret, for genetic differentiation and diversity in soil and plant pathogenic fungi, representing diverse species, order, and phyla. Pyret DNA markers showed the highest gene diversity and Shannon's information indices, followed by Maggy and Grasshopper. The observed high levels of multilocus polymorphism indicate the continuous mobility of these elements after their transfer in the new host. In conclusion, this study presents novel markers for genetic differentiation and evolutionary studies of fungi, and sheds light on the prevalence of gene acquisition phenomenon in field fungi.     

Cytotoxic and antibacterial activities of endophytic fungi isolated from plants at the National Park, Pahang, Malaysia

Background  

Endophytes, microorganisms which reside in plant tissues, have potential in producing novel metabolites for exploitation in medicine. Cytotoxic and antibacterial activities of a total of 300 endophytic fungi were investigated.     

Homologous linear plasmids in mitochondria of three species of wheat bunt fungi,Tilletia caries,T. laevis and T. controversa

Summary All isolates of Tilletia spp. investigated (five isolates of T. caries, including one from Japan, two isolates of T. laevis, and five isolates of T. controversa) contained a linear DNA plasmid ranging in size from 7.2 to 7.6 kb. All plasmids were highly homologous to each other as shown by DNA-DNA hybridization and comparison of restriction enzyme sites. Variability in the size of the plasmid was found to be due to differences within a central region of the plasmid. No homology between the plasmid and mitochondrial or nuclear DNA was found, but the mitochondrial origin of the plasmid was confirmed.     

Differentiation of species and strains among filamentous fungi by DNA fingerprinting

Summary We have analyzed 11 strains and clones, representing five species (Penicillium janthinellum, P. citrioviridae, P. chrysogenum, Aspergillus niger, Trichoderma harzianum) and three genera of filamentous fungi, for the presence of hypervariable loci in their genomes by hybridization with simple repeat oligonucleotides and the DNA of phage M13. The oligonucleotide probes (CT)₈, (GTG)₅ and (GACA)₄, as well as M13 DNA, are informative probes for fingerprinting in all genera and species tested. The probe (GATA)₄ produced informative fingerprints only with the genomic DNA of A. niger. There was no similarity between the fingerprints originating from fungi of different genera and also little similarity between the fingerprints of different species belonging to the same genus. Fingerprints of strains of the same species differed only slightly from each other. Fingerprints of clones originating from one strain were identical. The results indicate that DNA fingerprinting is a powerful method to differentiate species and strains of filamentous fungi.     

Prevalence and residential determinants of fungi within homes in Melbourne, Australia

BACKGROUND: Recent epidemiological studies suggest that the adverse respiratory health effects caused by the inhalation of fungal propagules are substantial. Knowledge of the prevalence and environmental determinants of indoor fungal levels is essential in designing effective avoidance measures. AIM: To investigate the prevalence of fungi and the influence of residential characteristics on levels of fungi within homes in Melbourne, Australia. METHODS: Floor dust and air samples were collected from bedrooms in 485 houses over 1 year. The dust was analysed for ergosterol, a marker of cumulative fungal biomass exposure. Total and genera-specific fungal propagules were identified in air samples. Details of the relevant residential characteristics were documented using a questionnaire. Independent predictors (P < 0.05) of ergosterol and total fungal propagules were identified by multiple linear regression. RESULTS: Fifty-five percent of the houses had viable fungal propagules exceeding 500 CFU/m3. Cladosporium and Penicillium were identified as the most prevalent and abundant fungal genera in indoor air. The median ergosterol level in bedroom floor was 3.8 microg/g of dust. Multivariate analysis showed that total fungal propagules in indoor air were lower in bedrooms with a ceiling fan, without visible mould, and those that were more frequently vacuumed, had a solid fuel fire, had windows closed at the time of the sampling or lacked pets. The presence of more than one cat had the greatest effect on total fungal propagules. Ergosterol levels were significantly lower in homes without old fitted carpets, visible mould or pets and those with frequent airing and regular use of an extractor fan in the kitchen. Old wall-to-wall carpets had the greatest effect on ergosterol. CONCLUSIONS: High indoor fungal exposures were associated with infrequent ventilation or vacuuming, presence of pets, visible mould and old carpets.     

Comparison of biochemical and immunologic properties of venoms from four hornet species

Venoms of two American hornets, Vespula maculata (bald-faced hornet) and Vespula arenaria (yellow hornet), and two Old World hornets, Vespa crabro and Vespa orientalis, were investigated for biochemical and immunologic properties. They were similar with regard to enzymatic activities (phospholipase A, phospholipase B, and hyaluronidase) and molecular composition. Analysis of radioallergosorbent test (RAST) results obtained from 30 vespid-sensitive patients suggests extensive cross-reactivity between the venoms of V. arenaria, V. maculata, and V. crabro, but the venom of V. orientalis seems to lack at least one important allergen. Rabbit antisera to each of the four venoms contain precipitating antibodies to all four venoms. Strong cross-reactivity was found between the venoms of V. maculata, V. arenaria, and V. crabro and between V. crabro and V. orientalis. V. orientalis venom cross-reacts weakly with the venoms of both V. maculata and V. arenaria. It is concluded therefore that diagnosis and immunotherapy with commercially available venoms of V. maculata and V. arenaria should be adequate for most patients sensitive to V. maculata, V. arenaria, or V. crabro but probably not for a significant proportion of those individuals primarily sensitive to V. orientalis.     

Immunoglobulin E-binding and skin test reactivity to hydrophobin HCh-1 from Cladosporium herbarum, the first allergenic cell wall component of fungi

BACKGROUND: For many years, fungal spores have been recognized as potential causes of respiratory allergies. All fungal allergens cloned so far represent either secreted or cytoplasmatic proteins, but nothing is known about the involvement of fungal surface proteins in allergic diseases. METHODS: A phage surface displayed cDNA-library from the mould Cladosporium herbarum was constructed and phage displaying IgE-binding proteins were selectively enriched with immobilized serum IgE from C. herbarum-sensitized individuals. Inserts encoding putative allergens were sequenced, subcloned and used to produce recombinant proteins. Allergenicity of the proteins was evaluated by IgE binding in Western blots, enzyme-linked immunosorbent assay (ELISA) and skin prick test in a total of 84 patients sensitized to either C. herbarum or Aspergillus fumigatus and three healthy controls. RESULTS: After four rounds of affinity selection, the cDNA-library was enriched for clones displaying IgE-binding molecules. Sequencing of inserts showed that one clone contained an open reading frame predicting a protein of 105 amino acids and a calculated molecular weight of 10.5 kDa showing the classical signature of members of the hydrophobin family. The recombinant protein, termed HCh-1, was able to bind IgE from six patients sensitized to fungi in vitro. Two of those patients were also included in a skin prick test survey and showed strong type I skin reactions to HCh-1, demonstrating the allergenic nature of C. herbarum hydrophobin and indicating a prevalence of sensitization in the range of 8-9%. In contrast, the hydrophobin HYP1 from Aspergillus fumigatus was not recognized by the sera of the same patients and controls investigated with HCh-1. CONCLUSION: C. herbarum hydrophobin represents the first component of the cell wall of fungi demonstrated to act as a rare but clinically relevant allergen in vitro and in vivo.     

Detection and species identification of Campylobacter in stool samples of children and animals from Vellore, south India

Campylobacter spp. are an important cause of bacterial gastroenteritis frequently isolated from animal, poultry and environmental samples. In this study, we investigated the zoonotic potential of Campylobacter spp. by comparing prevalence rates and species in 394 children with diarrhoea and 652 animals in Vellore using PCR-based tools. Eighteen children (4.5%) had campylobacteriosis, a majority of whom had co-pathogens (15/18) and most were infected with Campylobacter jejuni (16/18). A few C. coli and mixed infections with both species were also seen. Among the animal samples, 16/25 chicken samples (64%) were positive and all were found to be C. jejuni.     

Arbuscular mycorrhizal fungi and Trichoderma viride cooperative effect on biochemical,mineral content,and protein pattern of onion plants

The benefits of growth-stimulating microbes in crop production represent great opportunities for recent agricultural practices. Thus, the present investigation deals with examining whether arbuscular mycorrhizal (AM) fungi or Trichoderma viride application or their dual inoculation could improve the biochemical parameters and mineral and nutrient contents of onion plants (Allium cepa) under glasshouse conditions. The results evidenced that both AM fungi and T. viride are compatible with each other, and their combined use is effective, not only in improving the biochemical parameters, such as total soluble carbohydrates, protein contents, total free amino acids, acid, and alkaline phosphatases, but also in increasing mineral and nutrient contents (N, P, K⁺, Ca²⁺, Mg²⁺, and Zn) in onion plants, in which an increase of 67%, 49%, and 112% was observed in shoot onion P content with AM, T. viride, and with their dual inoculation, respectively, as compared with the controlled ones. Also, AM fungal colonization percentage augmented greatly with T. viride inoculation. Moreover, the protein profile of onion leaves revealed the appearance of newly protein bands with AM and T. viride applications. Therefore, their applications improved onion plant development, which could be used to replace the expensive chemical fertilizers, thus increasing onion quality.