Handbook Part 2 - International Mycological Association
Handbook Part 2 - International Mycological Association
Handbook Part 2 - International Mycological Association
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PS4-402-0238<br />
Lectin Accumulation in Edible Wild Mushrooms in Northeastern Thailand<br />
Sureelak Rodtong, Yubon Pikul-ngoen<br />
School of Microbiology, Institute of Science, Suranaree University of Technology, Nakhon Ratchasima 30000, Thailand<br />
Fungal lectins, the diverse multivalent carbohydrate-binding proteins of non-immune origin, have been becoming<br />
more interest due to the discovery of some of the lectins exhibiting antitumor activity as well as other potential<br />
activities such as mitogenic, immunoenhancing, and vasorelaxing activities. Some lectins derived from plants, are<br />
currently employed in a number of biomedical and clinical research. In Thailand, the great diversity of edible wild<br />
mushroom species has been reported. These mushrooms could provide an alternative source of lectins. In this study,<br />
the accumulation of lectins in fruit bodies of edible wild mushrooms found in Northeastern Thailand, was investigated.<br />
Some edible mushroom lectins may have cytotoxic activities against human cancer cells.<br />
Fresh fruit bodies of edible wild mushrooms were collected from their natural habitats and from local markets in<br />
Northeastern Thailand. The mushroom specimens were classified and identified using conventional methods based on<br />
their morphological characteristics, then dried, and ground into powder. Crude lectins were extracted from the<br />
powder, and detected their unique properties by hemagglutination assay using red blood cells from various animals<br />
(goose, guinea pig, mouse, rabbit, rat, and sheep). The extracts accumulating high lectin titers were selected to test<br />
for their temperature stability and cytotoxic activities against cancer cell lines, human epidermoid carcinoma (KB)<br />
and human cervical carcinoma (HeLa).<br />
From the 2-year collection of edible wild mushroom specimens, a total of 88 specimens with high morphological<br />
variation and belonging to the family Russulaceae, the dominant family found, were selected for crude lectin<br />
extraction. Sixty one specimens exhibited the incidence of lectin accumulation in their fruit bodies. The lectin extracts<br />
predominantly agglutinated rabbit and goose red blood cells, and performed their unique lectin properties<br />
depending on the mushroom strains. Crude extracts of six edible mushrooms in the genus Russula displaying their high<br />
lectin titres, were stable at 4 and 30ºC for 24 h, and at 60-70ºC for 30 min. Three of the six extracts still contained almost<br />
50% of lectin properties after exposing to 90ºC for 30 min. The six extracts exhibited different cytotoxic activities against<br />
KB and HeLa cell lines with IC50 values ranging from 16 to 170 and 16 to 700 mg/ml respectively. IC50 values less than<br />
30 mg/ml were designated as cytotoxicity.<br />
Specific strains of edible wild mushrooms in the family Russulaceae, particularly in the genus Russula, found in<br />
Northeastern Thailand, accumulated lectins in their fruit bodies. Some of the lectins showed their stability at high<br />
temperature, and could remain after cooking. Some also had cytotoxic activities against cancer, KB and HeLa, cell<br />
lines, which will be useful for further investigation.<br />
PS4-403-0239<br />
Estimation of fungal genome size using DAPI- image cytometry<br />
B. Kullman, W. Teterin<br />
1 Estonian University of Life Science, Tartu, Estonia, 2Rostock University, Rostock, Germany<br />
In providing quantitative data of nuclear DNA for the purpose of fungal taxonomy, photometric cytometry (PC) have<br />
played an important role. Fluorescence microscopy combined with computerised image analysis, i.e. image<br />
cytometry (IC), offers an alternative tool for assessing genome size. These techniques allow direct visualization of<br />
hyphae and simultaneous measurement of nuclear fluorescence intensity. We developed a simple method for<br />
quantitative evaluation of nuclear DNA in fungi using DAPI-IC. The intensity of signals from individual nuclei was<br />
quantitatively measured in digitized images. This simple IC performed on fruitbodies or on pure culture preparations<br />
enables to detect the amount of nuclear DNA in fungal cells.<br />
Staining Protocol . A slice of a fruitbody or a hypha of a pure culture were fixed in Carnoy’s solution and stored at 4<br />
°C until used, or at least for 1 h. To stain DNA, the fixed material was slightly dried and incubated with 0.5% Pepsin pH<br />
1.8 for 7 min at room temperature by slow shaking. Next, the fourfold volume of DAPI at 2˜g ml-1 TRIS buffer was<br />
added, and the sample was incubated for 45 min by slow shaking. Then the slices were placed in a drop of glycerin<br />
on glass slides, minced and rinsed gently with a shaving blade and squashed under the cover slips. The slides were<br />
stored at -20 °C. For one experiment, the slides of all specimens were prepared and analysed during one<br />
measurement session.<br />
Processing with Image Pro Plus 4.5 (manufactured by Media Cybernetics, USA)<br />
Hyphal nuclei were observed under 40x and 20x Olypmus LCPlan FL objectives and the images of the nuclei in the<br />
areas with low background noise were saved on the hard disk of the computer as TIFF files. Image-Pro Plus 4.5 was<br />
used to grab and process the images with local background determination: i.e. the nucleus was segmented<br />
determining the light intensity of the reference background from the narrow zone surrounding the nucleus using the<br />
cursor. Only a few nuclei in the centre of the image were measured because of the uneven illumination of the field of<br />
view. A rounded AOI with a stable size was used for selecting each nucleus separately throughout one measurement<br />
session. A total of 30-50 nuclei per slide were measured. If the parameter Integrated Optical Density (IOD) is selected<br />
with the Automatic Bright Objects option of the count/size command, then IOD is equal to Integrated Intensity of the<br />
nucleus to be measured.<br />
Proposed method. Image processing protocol, selection of the parameters and data collection: Menu Process?Color<br />
Channel?Extract: Color Model - RGB, Generate channel – B - OK. Menu Measure?Calibration, select Intensity: click<br />
New, Free Form, Options: Image, define a 3x3 neighborhood template, use the cursor as crosshairs to determine the<br />
Current Value of light intensity of the background for calibrating the input value ? OK, select Change to calibrate 0<br />
intensity for the y axis, Calibration always positive - OK. Menu Edit?New AO ?Select Ellipse AOI for measuring a nucleus<br />
- OK. Menu Edit>AOI: Add AOI with appropriate size, Save. Menu Measur?Count /Size, select Automatic Bright Objects,<br />
Measure Objects, Accumulate Counts, Display objects. Menu Measur e?Data Collector ?Layout: Count Size –<br />
selection: IOD; ?Data List?Collect Now after Count in Count /Size menu; ? Export?Select Excel (DDE)?Export Now. The<br />
research was supported from DAAD and by the ESF grant No. 4989.<br />
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