Calcofluors

In additional tests, the suppressor activities of pectins in the intact host/pathogen-interaction were investigated by injecting genetically resistant plants with pectic substances prior to inoculation with the rust fungus. Infected leaves were harvested, cleared, and stained with Calcofluor one week after inoculation, and fungal growth was assessed under the UV-epifluorescence microscope. 

The model in Figure 9 predicts that each microfibril would rotate in the process of cellulose ribbon formation. If the A. xylinum cell were held stationary, then the ribbon would be left-hand twisted (2-5) however, if the ribbon were held stationary, then the cell would rotate (32). The latter case explains why ribbons appear untwisted in the pellicle of ribbons shown in Figure 1. Moreover, it has been demonstrated that an A. xylinum cell ceased rotation when Calcofluor (> 0.1 mM) was added to the solution... 

Previous work has shown that the presence of Calcofluor or Tinopal could dramatically increase A. xylinum cellulose synthesis. This observation was the basis for the hypothesis that cellulose polymerization can be uncoupled from a slower sequential crystallization step (2-5). We believe the hypothesis is not consistent with our observations. At the very least, the presence of an ordered and crystal-like submicrofibril produced in the presence of 0.25 mM Tinopal would relegate Tinopal s or Calcofluor s effects to an event occurring after the initial cellulose polymerization-crystallization step or steps. 

Figure 3. Section of cooked rolled oat sample fixed in 3% glutaraldehyde, dehydrated and embedded in glycolmethacrylate. The stain is Calcofluor which is specific for P-Glucans in cell walls of cereals. Epifluorescence optics, (x 200).[From 26]...

Figure 15 Fluorescence intensity profiles of cross sections of three oat kernels with different levels of mixed-linkage beta-glucan after staining with the beta-glucan specific fluorochrome Calcofluor (highest peaks indicate highest concentrations of beta-glucan). Note that the polymer is not uniformly distributed throughout the kernel.

In some of the early experiments, organochlorine compounds were detected by fluorescence when the chromatogram was sprayed with N-methylcarbazole or rhoda-mine B C 1 33. Also, fluorescence whiteners (1 4) known as Calcofluors, were used to visualize carbamates, uracils, and ureas. Both of these techniques, however, were only of a semi-quantitative nature. 

Determination of the Calcofluor White Molar Extinction Coefficient Value in the Absence and Presence of -Acid Glycoprotein... 

Calcofluor White is commonly used to study the mechanism by which cellulose and other carbohydrate structures are formed in vivo and is also widely used in clinical studies (Andreas et al. 2000 Green et al 2000 Srinivasan 2004 Doctor Fungus website, Candida Endoph thalmitis). 

The interaction between Calcofluor White and carbohydrate residues of a -acid glycoprotein depends on the secondary structure of the carbohydrate residues, with the fluorescence parameters of Calcofluor being sensitive to this spatial secondary structure. 

When dissolved in water, the fluorescence maximum of Calcofluor White is 435-438 nm, while in alcohol such as isobutanol or when bound to human serum albumin, it fluoresces at 415 nm. In the presence of -acid glycoprotein, the fluorescence maximum of Calcofluor shifts toward 439 nm when the fluorophore is at low concentrations and toward 448 nm when it is present at high concentrations. The shift, compared to water and observed in the presence of a -acid glycoprotein, is the result of Calcofluor binding on the carbohydrates (40% by weight) of the protein (Albani and Plancke 1998,1999). 

To the sample cuvette, add with a pipette Pasteur very small quantity of Calcofluor White powder, then mix slowly. 

Plot absorption spectrum of Calcofluor White from 190 to 450 nm. 

Compare the absorption spectra for Calcofluor and oq -acid glycoprotein what do you notice Do you have identical spectra, and can you explain the result At which wavelength(s) do you have to determine the value of e of Calcofluor White, and why ... 

Determination ofe value of Calcofluor White free in PBS buffer... 

Prepare in PBS buffer a stock solution of Calcofluor White equal to 4.24 mg ml-1. To a cuvette containing 1 ml of buffer, add aliquots of 5 /xl from the Calcofluor White stock solution. After each addition, mix the solution slowly and measure the optical density at the wavelength you have chosen from the results you obtained in Section 2.3.1. Add at least 10 aliquots, and repeat the experiment twice. 

Once you finished the measurements, plot the optical density as a function of Calcofluor White concentration expressed in mg ml-1. What type of plot do you obtain, and why How do you expect to calculate the value of s from the plot you obtained The molecular weight of Calcofluor is equal to 942.7. Can you give the value of s in M-1 cm-1 ... 

Determination of Calcofluor White e value in the presence of oq -acid glycoprotein... 

Repeat the experiment described in Section 2.3.2 in the presence of 10 ptM of oq-acid glycoprotein (the e of the protein at 278 nm in PBS buffer, determined from the same method you are using to calculate the e of Calcofluor White, is equal to 29.7 mM-1 cm-1). 

Determination of Calcofluor White Molar Extinction Coefficient Value... 

Absorption spectrum of Calcofluor displays two peaks, one at 242 nm (clearly observed at high concentrations) and the second at 349 nm (Figure 2.4) We notice that both protein and Calcofluor absorb between 190 and 315 nm. At higher wavelengths, only Calcofluor absorbs, so in order to determine e for Calcofluor, one should work at wavelengths where Calcofluor only absorbs. The best wavelength will be at the peak equal to 349 nm. In the following experiment, we calculate the value of s at 352.7 nm. 

Plotting the optical density as a function of the Calcofluor concentration yields a linear plot with a slope equal to the product (Is) (Figure 2.5). For a cuvette path length l equal to 1 cm, the slope is equal to s, expressed in mg-1 1 cm-1. Multiplying the value of e by the protein s molecular weight yields an e expressed in mM-1 cm-1. The value of s calculated from the slope is equal to 4.65443 g-11 cm-1 or 4387.76 M-1 cm-1. 

In presence of 10 //M 04 -acid glycoprotein, a value of s equal to 4.15921 g-1 1 cm-1 or 3920 M-1 cm-1 is obtained. The value of e for bound Calcofluor White is around 9% lower than that of free Calcofluor in solution. 

Figure 2.5 Optical density plots of calcofluor white at 352.7 nm as a function of its concentration in (a) the absence and (b) the presence of 10 p.M -acid glycoprotein. In the absence of protein, s = 4.65443 g 1 I cm-1 = 4387.76 M 1 cm 1. In the presence of the protein, s = 4.15921 g-1 I cm 1 = 3920 M cm-1. Values of e are not the same the value of e for bound calcofluor white is around 9% lower than that of free calcofluor in solution.

Modification in the value of e is the result of structural reorganization in the vicinity of Calcofluor White. The presence of a protein alters the electronic distribution within the ligand, inducing a modification of absorption spectrum properties. 

Albani, J.R. and Plancke, Y.D. (1998 and 1999) Interaction between Calcofluor White and carbohydrates of ai-acid glycoprotein. Carbohydrate Research 314, 169-175 and 318, 194—200. 

Albani, J.R., Sillen, A., Coddeville, B., Plancke, Y.D. and Engelborghs, Y. (1999) Dynamics of carbohydrate residues of ai-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and emission anisotropy studies of Calcofluor White. Carbohydrate Research 322,87-94. 

Endophthalmitis.htm + clinical + studies, + Calcofluor + white hl = fr [accessed on 20 March, 2006]. 

Green, L.C., LeBlanc, P.J. and Didier, E.S. (2000) Discrimination between viable and dead encephalitozoon cuniculi (microsporidian) spores by dual staining with Sytox Green and Calcofluor White M2R. Journal of Clinical Microbiology 38, 3811-3814. 

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