Figure 1.
Thin-layer chromatography of silicic acid purified Cer fractions from astrocytes primary culture and from mouse brain lipid extract.
A. The Cer fractions (using previous method) from astrocytes were applied to the HPTLC plate and developed using chloroform:methanol:acetic acid (95:5:0.5, v/v/v). Ceramide bands were visualized after iodine absorption. Lanes 1–3: Std. NFACer; Lane 4–7: Ceramide fractions isolated from astrocytes; Lane 8: Cholesterol; Lane 9: Std. HFACer. Note: The appearance of contaminants (cholesterol) in all preparations. B. Pooled Cer fractions from astrocytes (1A) were applied on a silicic acid column and eluted with solvent choloroform;acetone:acetic acid (24:10.01, v/v/v, solvent 1). After eluting the column with 10 ml of the solvent 1, the column was eluted using choloroform;acetone:acetic acid (18∶2∶0.01, v/v/v, solvent 2). Approximately 1 ml fractions were collected and 10 µl was applied on the TLC plate and developed using chloroform:methanol:acetic acid (95∶5:0.5, v/v/v). The bands were visualized using char spray [6]. Lanes 1–2: Fractions collected using solvent 1 (approximately 5 ml each); Lanes 3–5, 7–12 fractions (1 ml each) collected using solvent 2; Lane 6: Std. NFACer. C. The purified Cer fractions (F2) from mouse brain were dissolved in chloroform:acetone 19:1. Fifteenµl was applied on each lane and bands were separated using chloroform:methanol:acetic acid (95:5:0.5, v/v/v) and visualized after iodine absorption as described in the text. Lanes 1–6: Ceramide fractions from brain; Lanes 7–9: Std. NFACer.
Table 1.
Fatty acyl composition of purified ceramide from astrocytes.
Figure 2.
GC-MS of TMS-bases of Cer purified from astrocytes.
The base composition of purified Cer was analyzed as TMS-derivative after N-acetylation as described in the text. A. TMS-Sphingosine base. B. TMS-Phytosphingosine base. Inserts indicate the primary m/z fragmentations.
Table 2.
Mass fragments (m/z) detected from TMS-base of ceramide purified from astrocytes, HOG cells and mouse brain.
Figure 3.
Thin-layer chromatography of purified Cer fractions and expression of DES2 in mouse tissues.
A. A defined amount of the purified Cer fraction (F2) from vertebrate tissues was spotted and resolved by HPTLC as described in the text. Samples were presented in duplicate. The plate was developed using chloroform:methanol;acetic acid 95:5:0.5 (v/v/v) and Cer bands were visualized after iodine absorption and char spray. Lane 1: Std. HFA-Cer; Lane 2–3: Brain; Lane 4–5: Liver; Lane 6–7: Kidney; Lane 8–9: Heart; Lane 10: Std. PHCer; Lane 11: Std. NFA-Cer. B. mRNA was prepared from mouse tissues and expression of DES2 determined by RT-PCR. Lanes 1 and 2: Brain; Lanes 3 and 4: Liver; Lanes 5 and 6: Heart.