The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)-D-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

cell wall, MALDIMS, β-glucan, Candida spp., limulus test

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](O)[C@H](*)O[C@H](CO)[C@H]1O

162.141 g/mol (C₆H₁₀R₂O₅, R = next and previous repeats, not counted in mol weight)

The limulus test is a well-established method for the diagnosis of both Gram-negative sepsis and invasive fungal infection. To diagnose fungal infections, a β-(1→3)-D-glucan-specific chromogenic kit (Fungitec G test MK) has been developed and applied clinically. We are concentrating our main efforts on developing a better standard to improve the precision of this method. To this end, we have successfully developed a protocol to obtain a soluble Candida spp. β-(1→3)-D-glucan (CSBG) by sodium hypochlorite (NaClO) oxidation and subsequent dimethyl sulfoxide (Me2SO) extraction (yield of 9.6 +/- 4.1%) of acetone-dried whole-cell preparations. The β-glucan fraction is free from the cell-wall mannan, gives a symmetrical peak by gel filtration, and is soluble in dilute NaOH. The product is composed mainly of β-(1→3)- and β-(1→6)-D-glucosidic linkages. The specific activity of the β-glucan is comparable with pachyman when combined with the Fungitec G test as the standard glucan and reacted as low as 10(-11) g/mL.

cell wall, MALDIMS, β-glucan, Candida spp., limulus test

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*OC[C@H]1O[C@@H](*)[C@H](O)[C@@H](O)[C@@H]1O

162.141 g/mol (C₆H₁₀R₂O₅, R = next and previous repeats, not counted in mol weight)

We previously reported that the fungal particle 1,3-β-D-glucan derived from Candida albicans, a pathogenic fungus, was obtained by oxidation of the cell wall with sodium hypochlorite (NaClO). It could be solubilized by treatment with dimethylsulfoxide (DMSO). In the present study, we prepared Candida 1,3-β-D-glucan having different physical properties, and examined the relationship between leukocyte activation and the physicochemical properties. β-glucan activated leukocytes significantly more effectively in a particulate than solubilized form in terms of TNF-α production by RAW 264.7 cells, hydrogen peroxide production by murine PEC and IL-8 production by human PBMC. Furthermore, we compared the biological activity of the glucan particles oxidized under various conditions. Interestingly, inactive and antagonistic particles were obtained under strong oxidation conditions. However, the inactive particles showed significant agonistic activity on dissolution in DMSO and following lyophilization. These facts strongly suggested that the solubility and assembly of the components influence the immunopharmacological activities of 1,3-β-D-glucans.

assembly, leukocyte, β-glucan, NaClO, DMSO

13C NMR data:
missing...

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

Warning = sub-repeat ending at bDGlcp: unknown unit count was assumed to be 3
*O[C@@H]1[C@@H](O)[C@H](*)O[C@H](CO[C@@H]2O[C@H](CO[C@@H]3O[C@H](CO[C@@H]4O[C@H](CO[C@@H]5O[C@H](CO)[C@@H](O)[C@H](O)[C@H]5O)[C@@H](O)[C@H](O)[C@H]4O)[C@@H](O)[C@H](O)[C@H]3O)[C@@H](O)[C@H](O)[C@H]2O)[C@H]1O

810.705 g/mol (C₃₀H₅₀R₂O₂₅, R = next and previous repeats, not counted in mol weight)

We previously reported that the fungal particle 1,3-β-D-glucan derived from Candida albicans, a pathogenic fungus, was obtained by oxidation of the cell wall with sodium hypochlorite (NaClO). It could be solubilized by treatment with dimethylsulfoxide (DMSO). In the present study, we prepared Candida 1,3-β-D-glucan having different physical properties, and examined the relationship between leukocyte activation and the physicochemical properties. β-glucan activated leukocytes significantly more effectively in a particulate than solubilized form in terms of TNF-α production by RAW 264.7 cells, hydrogen peroxide production by murine PEC and IL-8 production by human PBMC. Furthermore, we compared the biological activity of the glucan particles oxidized under various conditions. Interestingly, inactive and antagonistic particles were obtained under strong oxidation conditions. However, the inactive particles showed significant agonistic activity on dissolution in DMSO and following lyophilization. These facts strongly suggested that the solubility and assembly of the components influence the immunopharmacological activities of 1,3-β-D-glucans.

assembly, leukocyte, β-glucan, NaClO, DMSO

13C NMR data:
missing...

There is only one chemically distinct structure:

SVGMWSMILES3D molIonize

 

*O[C@@H]1[C@@H](O)[C@H](*)O[C@H](CO[C@@H]2O[C@H](CO)[C@@H](O)[C@H](O)[C@H]2O)[C@H]1O

324.282 g/mol (C₁₂H₂₀R₂O₁₀, R = next and previous repeats, not counted in mol weight)