Handbook Part 2 - International Mycological Association

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