Xi

1 234567 B 9

Fig. 1. Isolation of the proteoglycan Dally from third-instar Drosophila larvae by chromatography on DEAE Sepharose. Forty third-instar larvae were homogenized and fractionated on a DEAE column as described under Subheading 3.1. Thirty micrograms of protein from each fraction were separated by SDS-PAGE, blotted onto PVDF membrane (Millipore), and immunostained using a polyclonal anti-Dally antiserum at 1:15,000 dilution followed by goat anti-rabbit-HRP conjugate at 1:5000 and ECL (Amersham Pharmacia Biotech) according to the manufacturer's instructions. The lanes contain (1) flow-through, (2) column buffer (first wash), (3) column buffer (second wash), (4) low salt buffer, (5) 6 M urea, (6) pH 3.5 wash. (7) pH 8.0 wash, (8) 2 M NaCl (first wash), (9) 2 M NaCl (second wash). Dally elutes in the 2 M NaCl fractions as a smear between 90 and 200 kDa.

2. Materials

2.1. Purification of Proteoglycans from Drosophila

1. Homogenization buffer: 50 mM Tris HCl, 0.15 MNaCl, pH 8.0 1% CHAPS 2 x Complete Protease Inhibitors (Roche Molecular Biochemicals) (see Note 1). (6 M urea may be added to all buffers except the pH 8.0 wash and the high-salt buffer, or it may be added as a separate urea wash buffer [see Note 2]).

2. Column buffer: 50 mM Tris-HCl, 0.15 M NaCl, pH 8.0, 0.5% CHAPS, 1 x Complete Protease Inhibitors. (6 M urea: see Note 2.)

3. Low-salt buffer: 50 mM Tris-HCl, 0.25 M NaCl, pH 8.0, 0.1% Triton X-100, 1 x Complete Protease Inhibitors. (6 M urea: see Note 2.)

4. Urea wash buffer (used only if no urea is in the previous buffers [see Note 2]). 50 mM Tris-HCl, 0.25 M NaCl, pH 8.0, 6 M urea, 0.1% Triton X-100, 1 x Complete Protease

5. pH 3.5 buffer: 50 mM Sodium formate, 0.25 M NaCl, pH 3.5, 0.1% Triton X-100, 2 mM PMSF, 10 mM EDTA, 6 M urea. Urea is always included in this buffer (see Note 2).

6. pH 8.0 wash buffer : 50 mM Tris-HCl, pH 8.0, 0.5% CHAPS, 2 mM PMSF, 10 mM EDTA.

7. High-salt buffer: 50 mM Tris-HCl, 2 M NaCl, pH 8.0, 0.5% CHAPS, 2 mM PMSF, 10 mM EDTA.

8. A 0.5 x 3 cm chromatography column.

Inhibitors.

Fig. 2. Flow diagram of the postcolumn HPLC system used for analyzing the unsaturated disaccharides from Drosophila glycosaminoglycans. The sample is injected into the solvent pathway and separated on the column at 55° C with a NaCl gradient formed by the gradient pump. The column eluate is then mixed with 2-cyanoacetamide and NaOH, supplied by the double-plunger pump, and reacted at 125°C to form fluorescent products that, after cooling, are detected by the fluorescence detector. All solvents are degassed before they enter the separation and detection pathways. Solvent A is 1.2 mM ferf-ra-butylammonium hydrogen sulfate in 8.5% acetonitrile. Solvent B is 0.2 M NaCl in solvent A.

Fig. 2. Flow diagram of the postcolumn HPLC system used for analyzing the unsaturated disaccharides from Drosophila glycosaminoglycans. The sample is injected into the solvent pathway and separated on the column at 55° C with a NaCl gradient formed by the gradient pump. The column eluate is then mixed with 2-cyanoacetamide and NaOH, supplied by the double-plunger pump, and reacted at 125°C to form fluorescent products that, after cooling, are detected by the fluorescence detector. All solvents are degassed before they enter the separation and detection pathways. Solvent A is 1.2 mM ferf-ra-butylammonium hydrogen sulfate in 8.5% acetonitrile. Solvent B is 0.2 M NaCl in solvent A.

9. 0.3 mL of DEAE Sepharose Fast Flow (Amersham Pharmacia Biotech). (see Note 3).

10. A supply of Drosophila. For the volumes used in this chapter, initial experiments should be done with 40 pupae or third-instar larvae, 60 adults, 80 second-instar larvae, or 400 embryos or first-instar larvae. These numbers will have to be optimized for the needs of each individual experiment. (see Note 3).

2.1.1. For the Alternative Stepped NaCl Gradient Elution

1. Aliquots of column buffer (above) with total NaCl concentrations of 0.25, 0.5, 0.75, 1.0, 1.5, and 2.0 M. Because it is significantly less expensive, 0.1% Triton X-100 may be substituted for CHAPS in these buffers.

2.2. Purification of Proteoglycans from Drosophila S-2 Cells

1. CellFECTIN Reagent® (Life Technologies) (see Note 4).

2. Insect tissue culture medium. HQ-CCM3 serum-free insect cell culture medium (Hyclone) or Shields and Sang M-3 insect cell culture medium (Life Technologies, Sigma, or Hyclone) are both suitable. The latter must be supplemented with 12.5% fetal calf serum (Life Technologies, Sigma, or Hyclone) (see Note 5).

3. Transfection vector F449 (3) with the gene of interest under the control of the Hsp70 promoter. See Note 6.

4. To generate stable cell lines, a selection plasmid, such as pH8C0 (4) or pHGC0 (5), which confers methotrexate resistance (see Note 6).

5. Log-phase cultures of S-2 cells. S-2 cells are routinely maintained in dishes or flasks as nonadherent cells with a mean generation time of 24 h, and are split 1/5 every 5 days. To ensure optimum transfection rates, the cells should be split 1/1 in fresh medium the day before transfection. A thorough review of Drosophila cell culture can be found in Cherbas et al. (6).

Table 1

Disaccharide Standards from Chondroitin Sulfate

Growth medium containing 10-7 M methotrexate, if stable cell lines are being generated. TBS: 50 mM Tris-HCl, 0.15 M NaCl, pH 7.4, containing Complete Protease Inhibitor coctail (Roche Molecular Biochemicals).

2 mL of DEAE Sepharose Fast Flow (Amersham Pharmacia Biotech). A 1.4 x 4 cm glass chromatography column (Bio-Rad). A disposable polyproplyene column with a 3-mL capacity would also work. The column and buffer materials under Subheading 2.2.

2.3. Extraction of Glycosaminoglycans from Drosophila

GAG extraction solution: 0.5% SDS, 0.1 M NaOH, 0.8% NaBH4. 1.0 M sodium acetate. 1.0 M HCl. 80% Ethanol. Ethanol.

200- to 300-^m pore disposable filter column (Fisher Scientific). Drosophila or Drosophila tissue sample.

2.4. Microdetermination of Chondroitin Sulfate in Drosophila

1. Crude GAG solution (see Subheading 3.3.).

Digestion buffer: 0.2 M Tris-acetate buffer (pH 8.0). Chondroitinase ABC (E.C. 4.2.2.4) at 10 IU/mL (Seikagaku America). Chondroitinase ACII (E.C. 4.2.2.5) at 10 IU/mL (Seikagaku America). Unsaturated disaccharide standards from chondroitin sulfate (Seikagaku America). See Table 1.

2.5. Microdetermination of Heparan Sulfate in Drosophila

Crude GAG solution (see Subheading 3.3.).

Ultrafree MC Durapore microcentrifugal filtration units with 0.45 cm pores (Millipore). Ultrafree MC Biomax-5 microcentrifugal filtration units with a nominal molecular-weight exclusion of 5000 daltons (Millipore).

Table 2

Disaccharide Standards from Heparin/Heparan Sulfate

R1

R2

R3

1

AUA-GlcNAc

H

Ac

H

2

AUA-GlcNS

H

SOi

H

3

AUA-GI0NA06S

SOi

Ac

H

4

AUA2S-GlcNAc

H

Ac

SOi

5

AUA-GlcNS6S

SOi

SOi

H

6

AUA2S-GICNS

H

SOi

SOi

7

AUA2S-GlcNAc6S

SOi

Ac

SOi

8

AUA2S-GICNS6S

SOi

SOi

SOi

4. Ultrafree-MC DEAE microcentrifugal filtration units with DEAE-derivatized membranes (Millipore).

6. Loading buffer: 50 mM sodium phosphate buffer (pH 6.0) containing 0.15 M NaCl.

7. Elution buffer: loading buffer containing 1.0 M NaCl.

8. Heparin lyase mixture: 200 mlU/mL each of heparin lyase I (heparinase, E.C. 4.2.2.7), heparin lyase II (heparitinase II), heparin lyase III (heparitinase I, E.C. 4.2.2.8) (all from Seikagaku America).

9. Digestion buffer: 0.1 M sodium acetate buffer (pH 7.0) containing 10 mM calcium acetate.

10. Unsaturated disaccharide standards from heparin/heparan sulfate (Seikagaku America; see Table 2).

11. A Speed-Vac centrifugal evaporator (Savant).

2.6. HPLC Analysis

1. 1.2 mM tetra-ra-butylammonium hydrogen sulfate in 8.5 % acetonitrile.

2. 0.2 M NaCl in 1.2 mM tetra-ra-butylammonium hydrogen sulfate and 8.5% acetonitrile.

3. 0.5% 2-cyanoacetamide.

5. HPLC system shown in Fig, 2. For a detailed description of equiptment used by the authors, see Note 7.

3. Methods

3.1. Purification of Proteoglycans from Drosophila

1. Homogenize 40 third-instar larvae thoroughly in 3 mL of lysis buffer on ice, using 25-30 strokes with a Dounce homogenizer with the B pestle. One can also use a motorized pestle in a glass homogenizer. Pieces of adult fly cuticle and larval mouth parts will remain as black fragments in the homogenate, but the soft tissues should be completely dissociated. (see Note 1).

2. Centrifuge the lysate to clarify and remove excess lipid. To remove the major debris, centrifuge the lysate at 12,000g for 5 min at 4°C. Then recentrifuged supernatant at 30,000g for 30 min so that any lipid is removed from the surface of the clarified supernatant. (see Note 8).

3. Place 1 mL of DEAE Sephaeose suspension in a 15-mL conical centrifge tube and let the resin settle. Remove the overlying buffer and resuspend the resin in 10 mL of high-salt buffer. Equilibrate the resin with occasional mixing for 30 min, then let the resin settle and remove the buffer. Wash the resin twice with 10 mL column buffer by resuspension and settling.

4. The clarified supernatant is mixed with 0.3 mL of DEAE Sepharose (Amersham Pharmacia) and rocked at 4°C for 1 h (see Note 8).

5. The Sepharose resin is then allowed to settle and the overlying liquid is replaced with 1 mL of column buffer. Save the removed liquid (and all further fractions) on ice for analysis at the end of the column run.

6. The resin suspension is rocked for 5 min at 4°C, then allowed to settle and the overlying buffer is removed and saved as above. The resin is then resuspended in 1 mL of column buffer and packed into a 0.5 x 3 cm column. If the resin still contains dark flakes from the tanning reaction, repeat the washing step until the flakes are no longer visible to the naked eye. Then pack the column.

7. The column is washed successively with the following buffers. Collect fractions equivalent to 1 column volume:

a. 3 column volumes of column buffer.

b. 3 column volumes of low-salt buffer.

c. 3 column volumes of urea wash buffer (see Note 2).

d. 3 column volumes of pH 3.5 wash buffer. At this pH the carboxy side chains of Asp and Glu residues are protonated, thus releasing them from the column. Sulfates on GAG chains, however, remain negatively charged and bound to the column.

e. 4-5 column volumes of pH 8.0 wash buffer to return the pH of the column to 8.0. Check the pH of the 10 ^L effluent with 1 ^L of 0.1% phenol red. If it is still yellow, continue washing until the effluent is red-orange.

8. To elute the sulfated PGs from the column, add 1 column volume of high-salt buffer and let it drain to the surface of the resin. Close the column, add 1 column volume of buffer, and let it equilibrate for 20-30 min. Then reopen the column and collect the eluate. Close the column, and repeat the elution step with 1 column volume of high-salt buffer. Then wash with the remaining high-salt buffer without closing the column (see Note 9).

9. Store fractions on ice, and assay 10 ^L of each fraction for protein content using the BCA or other suitable protein assay.

10. Samples can then be dialyzed, precipitated, and assayed by PAGE or other means. (see Fig. 2).

3.1.2. Alternative Stepped NaCl Gradient Elution

1. Follow the standard procedure through step 7b.

2. Wash the column successively with 2-mL aliquots of column buffer containing 0.25, 0.5, 0.75, 1.0, and 1.5 M NaCl.

3. Elute the most tightly bound material from the column with 3 mL of buffer with 2.0 M NaCl.

4. Dialyze and analyze fractions as for the standard protocol.

3.2. DEAE Isolation of Sulfated Proteoglycans from Drosophila S-2 cells

3.2.1. Transfection of Drosophila S-2 Cells

1. Add 2 ^g each of expression vector and selection plasmid DNA to be transfected and 10 ^g CellFECTIN reagent to 0.5 mL of serum-free medium, vortex briefly, and incubate at room temperature for 20-40 min. (see Note 6).

2. Resuspend 2 x 106 S-2 cells in mid-log phase in 0.5 mL of serum-free medium. (see Note 5).

3. Gently mix the DNA/CellFECTIN solution with the cell suspension and plate in a 35 mm-diameter culture dish or in a well of a 6-well culture plate and incubate at 23 °C for 4-6 h.

4. Add 1 mL of fresh growth medium to the cells and return to the incubator overnight.

5. In the morning, add 1 mL of fresh growth medium to the cells and return to the incubator for 7-8 h.

6. Gently resuspend the cell pellets by centrifugation at 300g for 5 min. Then wash the cells once by resuspending and repelleting in serum-free medium to remove excess CellFECTIN reagent and DNA.

7. Replate the cells in 3 mL of fresh growth medium. If stable cell lines are to be created, add the selection medium at this time. Use 2 x 10-7 M methotrexate for cells co-transfected with the selection plasmid pH8 C0.

8. Transient transfectants can be grown for 12-48 h before use. Stable transfectants are grown for 1 wk. Aliquots are then frozen in liquid nitrogen.

3.2.2. Harvesting Cells and Fractionation PGs

1. Heat-shock 3 x 109 log-phase S-2 cells transfected with the desired gene cloned into F449 or another suitable vector (see Note 6): 30 min at 37°C, 5 min on ice, 2-4 h recovery at 23°C.

2. Resuspend nonattached cells and put in a centrifuge tube on ice.

3. Remove attached cells by scraping in 2 x 5 mL of ice-cold TBS, pH 7.4, containing protease inhibitors, and add to centrifuge tube.

Carry out the following work at 4°C.

4. Spin cells for 5 min at 3000g, remove the supernatant, and wash the cells twice with 10 mL of TBS.

5. Homogenize the cells in 10 mL of homogenization buffer, on ice, 3 min, with a motorized Teflon pestle in a glass homogenizer.

6. Centrifuge homogenate at 12,000g.

7. Place 6 mL of DEAE Sephaeose suspension in a 50-mL conical centrifge tube and let the resin settle. Remove the overlying buffer and resuspend the resin in 40 mL of high-salt buffer. Equilibrate the resin with occasional mixing for 30 min, then let the resin settle and remove the buffer. Wash the resin twice with 40 mL of column buffer by resuspension and settling.

8. Apply supernatant to a 2-mL DEAE Sepharose column and elute with the protocol described for whole animal lysates (see Subheading 3.1.).

9. Dialyze aliquots of each fraction and analyze by PAGE/Western blotting for the presence of the desired core proteins.

10. Store the remaining fractions at -20°C.

3.3. Extraction of Glycosaminoglycans from Drosophila

1. To extract GAGs from Drosophila, lyophilize 100 adult flies, 100 larvae or 250 ^L of dechorionated embryos, or wash with distilled water and then lyophilize.

Retention tjme (min)

2. Homogenize samples (up to 20 mg of lyophilized samples) in 1.0 mL of acetone cooled on ice. Extract for 30 min on ice, then centrifuge 15 min at 2500g at 4°C. Wash the precipitate 3 times with 1.0 mL of ice-cold acetone and dry under vacuum.

3. Extract samples in 1.0 mL of GAG extraction solution for 16 h at room temperature with constant stirring.

4. Neutralize samples with 200 ^L of 1.0 M sodium acetate and 300 ^L of 1.0 M HCl and centrifuge 10 min at 2500g, 4°C. Remove particulate matter from the supernatant with a 300-^m-pore disposable filter column.

5. Add 200 ^L of 1.0 M HCl to the filtrate and remove insoluble materials by centrifugation for 10 min at 2500g, 4°C, then add 7 mL of ethanol to the supernatant and allow the GAGs to precipitate for 2 h at 0°C.

6. Centrifuge 10 min at 2500g at 4°C and remove supernatant. Wash the precipitates once with 1 mL of 80% ethanol and once with 1 mL of ethanol. These washes should be done at 4° C. Dry the precipitate under vacuum. At this stage the samples can be stored at -20°C until needed.

7. For use in determining the chondroitin sulfate and heparan sulfate disaccharide ratios (following protocols), dissolve the crude GAG pellets in 250 ^L of distilled water.

3.4. Microdetermination of Chondroitin Sulfate in Drosophila

1. Dilute 20 ^L of crude GAG solution to 100 ^L with distilled water.

2. Add 10 ^L of digestion buffer, 5 ^L of chondroitinase ABC solution, and 5 ^L of chondroitinase ACII solution to 20 ^L of the diluted crude GAG solution in a 500-^L polypropylene microcentrifuge tube, then incubate at 37°C for 3 h.

3. Spin down the tube in an Eppendorff centrifuge for 30 s, then heat the tube for 2 min at 100°C and spin down again.

4. Submit 8 ^L of the digest to HPLC analysis using the method under Subheading 3.6. Use 2 ppm of each unsaturated disaccharide mixture as standard. A typical chromatogram is shown in Fig. 3A.

3.5. Microdetermination of Heparan Sulfate in Drosophila

1. Add 400 ^L of elution buffer to the Ultrafree-MC DEAE insert and spin at 5000g for 1 min.

2. Empty the microcentrifuge tube. Then add 400 ^L of loading buffer to the insert and spin at 5000g for 1 min. Transfer the insert to a new microcentrifuge tube.

3. Add 50 ^L of 0.3 M sodium phosphate buffer (pH 6.0) to 230 ^L of crude GAG solution.

4. Filter the mixture through a Ultrafree MC (Durapore®, 0.45 ^m).

5. Add the filtrate to the Ultrafree-MC DEAE insert and spin at 5000g for 1 min. Pass the sample over the membrane twice.

6. Transfer the insert to a new microcentrifuge tube. Add 400 ^L of loading buffer to the insert and spin at 5000g for 1 min.

7. Transfer the insert to a new microcentrifuge tube. Add 100 ^L of elution buffer to the insert and spin at 5000g for 1 min. Repeat 3 times. This fraction is the eluate.

Fig. 3. HPLC profiles of CS and HS disaccharides. Typical chromatograms of unsaturated disaccharides from chondroitin sulfate (A) and heparan sulfate (B) from adult Drosophila. The positions at which disaccharide standards migrate are indicated by the arrows. The numbers above the standards correspond to the numbers of their respective elution peaks. It is interesting to note that the only sulfated chondroitin disaccharide detected in Drosophila is ADi-4S.

8. Add the eluate to a Ultrafree MC (Biomax-5) and spin until the retentate is 30 ^L.

9. Add 50 ^L of distilled water to the retentate and spin. Repeat four times.

10. Remove the retentate to a new tube with a pipette, and rinse the membrane four times with 20 ^L of distilled water. Add the washes to the retentate.

11. Dry the sample in a centrifugal evaporator, then dissolve it in 12 ^L of distilled water.

12. Add 5 ^L of digestion buffer and 5 ^L of heparin lyase mixture to 5 ^L of the partial purified heparan sulfate from step 11 in a 500-^L polypropylene microcentrifuge tube, then incubate at 37°C for 16 h.

13. Centrifuge for 30 s at 12,000g, then heat the tube for 2 min at 100°C and recentrifuge.

14. Dry the digest in a centrifugal evaporator.

15. Add 10 ^L of water, then inject 8 ^L of the sample into the HPLC for analysis using the method under Subheading 3.6. Use 5 ppm of each unsaturated disaccharide mixture as standard. A typical chromatogram is shown in Fig. 3B.

3.6. HPLC Analysis

A flow diagram of the HPLC system is shown in Fig. 2. For a detailed description of the HPLC system used in this method, (see Note 9).

1. Before injecting the sample, heat the column to 55°C and equilibrate with 1.2 mM tetra-ra-butylammonium hydrogen sulfate and 2 mM NaCl in 8.5% acetonitrile. Warm the postcolumn reaction coil to 125°C.

2. Inject the disaccharides and elute at a flow rate of 1.1 mL/min with an NaCl gradient consisting of the following segments:

All gradient components contain 1.2 mM tetra-ra-butylammonium hydrogen sulfate and 8.5% acetonitrile. The column is then reequilibrated with 1% 0.2 M sodium chloride for 20 min.

3. To detect the eluted disaccharides, aqueous 0.5% (w/v) 2-cyanoacetamide solution and 1% (w/v) sodium hydroxide are added to the column effluent at the flow rate of 0.35 mL/min. The mixture is then passed through the reaction coil at 125°C, and following cooling, the effluent is monitored fluorometrically at 410 nm with an excitation wavelength of 346 nm.

4. Notes

1. Drosophila larvae and pupae can have high levels of protease activity. It is therefore imperative that all work be done in the cold and in the presence of a complete cocktail of protease inhibitors. A mixture of protease inhibitors that inhibit serine-, cysteine-, and metalloproteases must be included in the lysis buffer and the first 2-3 column wash buffers. The remaining buffers should contain at least 2 mM PMSF and 10 mM EDTA. The inclusion of these inhibitors will help prevent the degradation of the core proteins of the proteoglycans being isolated. Several manufacturers supply suitable protease mixtures in liquid or tablet form.

2. To help solubilize proteoglycan, 6 M urea may be added to the homogenization buffer. In that case it should be included in all buffers except the pH 8.0 wash buffer and the high salt buffer. Alternately, 6 M urea may be used as a separate wash step (urea buffer) to help dissociate protein aggregates from the column. Urea should always be included in the pH 3.5 buffer. It should be noted that urea in solution readily oxidizes to uric acid. Thus, all urea-containing solutions should be prepared from the highest-quality urea available and just prior to use. Including urea in the homogenization buffer may increase the yield of proteoglycans, but it denatures the core protein and must be removed if the samples are to be subjected to digestion by GAG lyases.

3. DEAE Sepharose Fast Flow has a maximum binding capacity of 0.11-0.16 mmol/mL resin, which is equivalent to 3-4 mg of a 90-kDa protein or proteoglycan. Thus, a 0.3 mL column has a theoretical capacity for PGs from several hundred third-instar larvae.

4. Calcium phosphate has also been used extensively to transfect S-2 cells, and electroporation has been used with Drosophila KC167 cells. These methods are well reviewed by Cherbas et al. (6).

5. Drosophila cells can be maintained in a number of media. Most commonly used for S-2 cells are M3 (7), D-22 (8), and Schneider's medium (9), all of which are commercially available (Sigma, Life Technologies) and must be supplemented with 5-12.5% fetal bovine serum. Purchase fetal bovine serum that has been tested for use with tissue culture cells, because not all lots of serum support insect cell growth. Hyclone's HQ-CCM3 is a serumfree medium that works well with S-2 cells and, lacking the need for serum supplementation, is less expensive than the other media. Cherbas et al. (6) report that HQ-CCM3 is also preferable to M3 for methotrexate and G418 selection.

6. The most commonly used expression vectors used in Drosophila cells put the gene of interest under the control of promoters for HSP 70, metallothionein, or ecdysone (reviewed by Cherbas et al. [6]). Since S-2 cells take up several hundred to several thousand copies of the transfected DNA, it is not necessary to have the resistance gene used to generate stable cell lines on the same plasmid as the gene of interest. In all but a few exceptional cases, co-transfecting a selection plasmid which carries a selectable resistance gene with the gene of interest in a separate expression vector provides stable cell lines expressing the desired gene product. Methods using selection plasmids carrying resistance to methotrexate, G418, and hygromycin can be found in Ashburner (10).

7. The chromatographic equipment used by the authors includes an L-7000 gradient pump and D-7500 chromato-integrator (Hitachi Instruments) and a model 7125 sample injector with a 20-^L loop (Rheodyne). Samples were separated on a 4.6 mm x 150 mm Senshu Pak DOCOSIL A column (Senshu Scientific, Tokyo, Japan).The postcolumn reaction system consists of a double-plunger pump, AA-100-S (Eldex Laboratories), a CH-30 column heater, a FH-40 dry reaction bath, and a TC-55 thermocontroller (Brinkmann Instruments). Samples are detected with a RF-10AXL fluorescence spectrophotometer (SHIMADZU SCIENTIFIC).

8. The wound-healing response of Drosophila and other insects involves a rapid tanning reaction that converts components in the hemolymph into dark, cuticle-like material. When Drosophila are homogenized, this reaction can produce a fine suspension of particles that are not always removed by centrifugation at modest speeds. These particles can plug columns and disrupt biochemical fractionation. Two rounds of centrifugation, followed by loading and washing the ion-exchange resin in batches, allows the particles to be washed away before the resin is packed into a column.

9. By washing 1 column volume of elution buffer through the column, then closing the column and letting the resin equilibrate for 20-30 min with 1 column volume of fresh buffer, most of the bound ligand will be released into the second elution buffer fraction.

10. The most practical quantitative approach to the analysis of the unsaturated disaccharides derived enzymatically from GAGs is detection of their ultarviolet absorption at 232 nm. However, detection systems are not sensitive enough for the microdetermination of biologi-

cal samples. To improve the detection limits and specificity, pre- and postcolumn detection techniques have been investigated. The postcolumn method using 2-cyanoacetamide (11) was specifically developed to detect optimally unsaturated disaccharides from small amounts of Drosophila GAGs.

References

1. Herndon, M. E. and Lander, A. D. (1990). Diverse set of developmentally regulated proteoglycans is expressed in the rat central nervous system. Neuron 4, 949-961.

2. Toyoda, H., Kinoshita-Toyoda, A., and Selleck, S. B. (2000). Structural analysis of glycosoaminoglycans in Drosophila and C. elegans and demonstration that tout-velu, a Drosophila gene related to EXT tumor suppressors, affects heparan sulfate in vivo. J. Biol. Chem. 275, 2269-2275.

3. Struhl, G. (1985). Near-reciprocal phenotypes caused by inactivation or indiscriminate expression of the Drosophila segmentation gene ftz. Nature 318, 677-80.

4. Rebay, I., Fleming, R. J., Fehon, R. G., Cherbas, L., Cherbas, P., and Artavanis-Tsakonas, S. (1991). Specific EGF repeats of Notch mediate interactions with Delta and Serrate: implications for Notch as a multifunctional receptor. Cell 67, 687-699.

5. Bunch, T. A. and Goldstein, L. S. (1989). The conditional inhibition of gene expression in cultured Drosophila cells by antisense RNA. Nucleic Acids Res. 17(23), 9761-9782.

6. Cherbas, L., Moss, R., and Cherbas, P. (1994). Transformation techniques for Drosophila cell lines. Meth. Cell Biol 44, 161-179.

7. Shields, G. and Sang, J. H. (1977). Improved medium for the culture of Drosophila embryonic cells. Drosophila Information Service 52, 161.

8. Echalier, G. and Ohanessian, A. (1970). In vitro culture of Drosophila melanogaster embryonic cells. In Vitro 6, 162-172.

9. Schneider, I. (1964). Differentiation of larval Drosophila eye-antennal discs in vitro. J. Exp. Zool. 156, 91-103.

10. Ashburner, M. (1989). Drosophila: A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY.

11. Toyoda, H., Yamamoto, H., Ogino, N., Tioda, T.,and Imanari, T. (1999). Rapid and sensitive analysis of disaccharide composition in heparin and heparan sulfate by reversed-phase ion pair chromatography on 2 ^m porous silica gel column. J. Chromatogr. A 830, 197-201.

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