- Email: [email protected]
Solid-Phase Extraction of Lipid from Saccharomyces cerevisiae Followed by High-Performance Liquid Chromatography Analysis of Coenzyme Q Content
NOTES & TIPS 3. Grunwald, A. G. (2000) Characterization and sequence analysis of the toluene ortho-monooxygenase of Burkholderia cepacia G4, M.S. Thesis, Univ. West Florida. 4. Horn, N. A., Meek, J. A., Budahazi, G., and Marquet, M. (1995) Cancer gene therapy using plasmid DNA: Purification of DNA for human clinical trials. Hum. Gene Ther. 6, 565–573. 5. Levison, P. R., Badger, S., Hathi, P., Davies, M., Bruce, I., and Grimm, V. (1998) New approaches to the isolation of DNA by ion-exchange chromatography. J. Chromatogr. A 827, 337–344. 6. Moreau, N., Tabary, X., and LeGoffie, F. (1987) Purification and separation of various plasmid forms by exclusion chromatography. Anal. Biochem. 166, 188 –193. 7. Prazeres, D. M. F., Guilherme, N. M., Monteiro, G. A., Cooney, C. L., and Cabral, J. M. S. (1999) Large-scale production of pharmaceutical-grade plasmid DNA for gene therapy: Problems and bottlenecks. Trends Biotechnol. 17, 169 –174. 8. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 9. Shields, M., Reagin, M., Gerger, R., Campbell, R., and Somerville, C. (1995) TOM: A new aromatic degradative plasmid from Burkholderia (Pseudomonas) cepacia G4. Appl. Environ. Microbiol. 61, 1352–1356. 10. Yannish-Perron, C., Viera, J., and Messing, J. (1985) Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103–119.
Solid-Phase Extraction of Lipid from Saccharomyces cerevisiae Followed by High-Performance Liquid Chromatography Analysis of Coenzyme Q Content Ruth A. Hagerman, Mark J. Anthony, and Richard A. Willis 1 Division of Nutritional Sciences, University of Texas, Austin, Texas 78712 Received April 6, 2001; published online August 6, 2001
Ubiquinone (coenzyme Q; Q) 2 functions in the mitochondria to transport electrons between complexes I and III of the electron transport chain, and to accept electrons from succinate. All eukaryotic organisms that undergo respiration produce Q, although the specific type of Q varies with the organism. Humans produce Q 10, containing a 10-unit isoprenoid side chain, whereas Saccharomyces cerevisiae (baker’s yeast) produces Q 6. The utility of yeast as a model eukaryote is of interest to many laboratories studying coenzyme Q, but no standard assay of yeast Q 6 content has been established. In this paper, a simple method of lipid extraction followed by solid-phase purification and 1
To whom correspondence should be addressed at Division of Nutritional Sciences, Mail Code A2703, University of Texas at Austin, Austin, TX 78712. Fax: (512) 471-7784. 2 Abbreviations used: Q, coenzyme Q. Analytical Biochemistry 296, 141–143 (2001) doi:10.1006/abio.2001.5224 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
141
HPLC analysis is described. This method was developed to remove the majority of contaminants such as ergosterol from lipid extracts, thus allowing a larger quantity of Q 6 to be analyzed by HPLC. In addition, this method reduces HPLC run times from 70 to 15 min, leading to significant savings of time and solvents. Data generated using this method are presented. Several methods of isolating Q 6 from yeast have been reported. Crane and Barr (1) suggested methanolic saponification to remove ergosterol from acetone extracts of yeast, but reported that yields of Q 6 were much reduced after saponification. Santos-Ocana et al. (2) improved the saponification procedure, but the method required large volumes of solvent. Poon et al. (3) described a sorptive method for removal of ergosterol, which required a second lipid extraction with resultant losses of Q 6 recovery and time. Schultz and Clarke (4) extracted Q 6 from yeast using methanol: petroleum ether. In this method, the yeast cells were dried for 24 h at 80°C, lysed, and extracted. This method was modified slightly by the same laboratory the following year (5). Modifications included drying the cells for 48 h at 37°C, and the use of Q 9 as an internal standard to correct for lipid losses during extraction. However, neither method removes ergosterol from the samples before analysis. We modified the methods of Schultz and Clarke (4) and Jonassen and Clarke (5) as follows. Yeast were grown for 24 h in treatment media and cell pellets were dried under a stream of nitrogen for 2 h at 56°C. Pellet weights obtained from drying under nitrogen were compared to drying for 48 h at 37°C (5) and found to be essentially identical (1.41 and 1.48 mg, respectively). Dry pellets were resuspended in 0.5 ml water, glass beads (0.5 mm) and 1 g of Q 7 (Sigma; from a stock solution of 20 g/ml ethanol) were added, and the suspension was vortexed for 2 min. Ten milliliters of methanol:petroleum ether (6:4) was added, the mixture was vortexed briefly, and 1 ml of water was added to achieve proper phase separation. In order to achieve clear separation of phases, the proportions must be at least 1:6:4 (water:methanol:petroleum ether, v/v), although more water can be added (we used 1.5:6:4). Thus, cell pellets of any weight suspended in a convenient volume of water may be extracted using these proportions. Phases were separated by centrifugation (1000g) and the organic phase (top layer) was reserved. The pellet and lower phase were reextracted once with 4 ml of petroleum ether. Crane and Barr (1) suggested that the lipid extraction should be repeated sequentially to verify that the maximum amount of Q 6 is obtained. In our hands, a representative sequential extraction yielded 164.1 g Q 6/g dry weight of cells in the first extraction, followed by 15.8 g/g in the second, and 1.3 g/g in the third. Thus, our method includes only two extractions
142
NOTES & TIPS
FIG. 1. HPLC analysis of lipid extracted from yeast. Lipids were extracted from yeast and analyzed for Q 6 content (A) as described in the text. An additional solid-phase extraction step removed approximately 96% of the ergosterol (B).
of the pellet. Supernatants were pooled, dried under nitrogen, and stored at 4°C. In Fig. 1A, a representative chromatogram of the crude unfiltered extract is presented. Lipid samples were dried completely under nitrogen and resuspended in a known volume of isopropanol. Samples were analyzed on a Beckman 168 HPLC with a diode array detector using System Gold Nouveau software and a Zorbax ODS reversed-phase column. Compounds were separated using an isocratic mobile phase of hexane: methanol (1:9, v/v), at a flow rate of 1.2 ml/min. Both Q 6 and Q 7 were detected at 275 nm. No peak was visible at 290 nm, the characteristic absorbance region of reduced CoQ. The identities of ergosterol, Q 6, and Q 7 were confirmed by mass spectrometry (Department of Chemistry, The University of Texas at Austin). Under these conditions, the ergosterol peak at 5.3 min (and a derivative at 5.8 min, also seen in the
standard purchased from Sigma) often obscured the Q 6 peak, making analysis difficult. There also were four major compounds eluting late in the run (43.6, 50.8, 58.5, and 67.9 min; data not shown) which necessitated a total run length of 70 min. These peaks were collected from the HPLC, dried, and submitted for identification by mass spectrometry (Department of Chemistry, University of Texas at Austin). A positive identification was not obtained; however, based on the UV absorbance spectra and mass analysis, we believe these compounds are precursors or derivatives of ergosterol. We developed a method using solid-phase extraction to remove the 4 late peaks and ergosterol from the lipid extract before HPLC analysis. In Fig. 1B, a representative chromatogram from a column-treated sample is shown. A 3-cm hand-packed column of 230 – 400 mesh silica gel (Sigma) was prepared in a 5-inch glass pipette and washed with 3 ml of dichloromethane:hexane. The silica was not heat-activated prior to packing the column. The dried lipid extract (5–10 g) was dissolved in 1 ml of dichloromethane:hexane (2.5:1, v/v) and applied to the column. An additional 1 ml of dichloromethane:hexane was added and the eluate collected in 0.5-ml fractions. As shown in Table 1, the majority of the two largest peaks eluted in the first 2 ml. Peaks III and IV also elute in these fractions (data not shown). The next 2 ml of dichloromethane:hexane contained the majority of the Q 6 and Q 7. Approximately 96% of the ergosterol remained on the column and can be eluted with an additional 3 ml of 100% dichloromethane. Thus, for Q 6 analysis, fractions 5 through 8 can be reserved for HPLC analysis and the remainder discarded. HPLC analysis is as described above but run times are cut from 70 to 15 min. Traces of contaminants may remain in the sample after the solid-phase extraction. When run times for Q 6 analysis are reduced to 15 min, a routine 70-min flush of the
TABLE 1
Elution Profile of Solid-Phase Extraction Column a Fraction b (0.5 ml)
Peak I
Peak II
CoQ 6
CoQ 7
Ergosterol
1 2 3 4 5 6 7 8 9 10
306962 1953593 374433 44454 — — — — — —
66073 315246 74462 5354 — — — — — —
— — — 42142 73842 91900 53300 38089 — —
— — — 11577 20873 23056 9993 3579 — —
— — — — — — — — — 5290344
Data are area under the curve from a 5 g sample of lipid. Fractions 1 to 9 are in units of 0.5 ml of dichloromethane:hexane (2.5:1, v/v). Fraction 10 is a wash of 3 ml of dichloromethane. a b
143
NOTES & TIPS
yeast. In addition, we found that cell drying times could be reduced from 2 days to 2 h. Using this method, we have shown that yeast grown on ␥-linolenic acid for 24 h produce less Q 6 than yeast grown on the unsaturated fatty acids, oleic, elaidic, or linoleic, although cells grew to the same final concentration in all media. Acknowledgments. The authors thank Dr. Ann E. Hagerman for critical reading of this manuscript. This research was supported in part by the Karl Folkers Foundation.
REFERENCES FIG. 2. Yeast grown on different fatty acids produce different amounts of Q 6. Yeast were grown for 24 h in the indicated fatty acids and analyzed for Q 6 content as described in the text. Data are expressed as microgram Q 6 per gram dry weight of cells (mean ⫾ SD, n ⫽ 3 to 5).
system with a blank sample is included approximately every 10 runs to remove any trace compounds which may have accumulated on the column. Removal of the majority of the ergosterol from the chromatograms improved quantitation of Q 6 significantly. Some analyte was lost in the solid-phase extraction step, but inclusion of the internal standard (Q 7) provided a convenient method for correcting that loss. Using this method, approximately 30% of the internal standard was lost, compared to typical losses of 2 to 12% in crude extractions. We have used this method successfully to analyze the Q 6 content of yeast grown on different fatty acids. The yeast strain SEY6210 was grown at 30°C while shaking in defined yeast medium (6% yeast nitrogen base, 0.5% yeast extract, 1% tergitol NP-40, and amino acids) containing 1 mM fatty acid (oleic, elaidic, linoleic, or ␥-linolenic). Yeast were harvested at 24 h and Q 6 analyzed as described above. Figure 2 shows that yeast grown on the -9 fatty acids oleic (cis 18:1) or elaidic (trans 18:1) produced the most Q 6 (15.9 ⫾ 2.5 and 18.3 ⫾ 1.6 g/g dry weight of cells, respectively). Slightly less Q 6 was produced after growth on linoleic (cis, cis 18:2, -6; 12.2 ⫾ 2.5 g/g dry weight) and much less after growth on ␥-linolenic (all cis 18:3, -6; 7.3 ⫾ 0.7 g/g dry weight). To compare growth rates, absorbance at 600 nm was monitored. At 24 h, all carbon sources allowed for the growth of cells to the same concentration, although the cells grew at different rates (data not shown). Thus, the differences in cellular Q 6 content were not reflected in cell growth. In conclusion, solid-phase extraction of lipid from yeast yields a fraction containing only 4% of the initial ergosterol. The additional major lipid contaminants are also no longer present. The extracted samples yield cleaner chromatograms and require run times of only 15 min, resulting in significant savings of time and volume of solvent required for analysis of Q 6 from
1. Crane, F. L., and Barr, R. (1971) Determination of ubiquinones. Methods Enzymol. 18, 137–165. 2. Santos-Ocana, C., Cordoba, F., Crane, F. L., Clarke, C. F., and Navas, P. (1998) Coenzyme Q 6 and iron reduction are responsible for the extracellular ascorbate stabilization at the plasma membrane of Saccharomyces cerevisiae. J. Biol. Chem. 273, 8099 – 8105. 3. Poon, W. W., Marbois, B. N., Faull, K. F., and Clarke, C. F. (1995) 3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 320, 305–314. 4. Schultz, J. R., and Clarke, C. F. (1999) Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants. Biofactors 9, 121–129. 5. Jonassen, T., and Clarke, C. F. (2000) Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis. J. Biol. Chem. 275, 12381– 12387.
High Transfection Efficiency of Human Umbilical Vein Endothelial Cells Using an Optimized Calcium Phosphate Method Inmaculada Segura, Manuel A. Gonza´lez, Antonio Serrano, Jose´ Luis Abad, Antonio Bernad, and Hans H. Riese 1 Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas/Universidad Auto´noma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Received April 12, 2001; published online August 3, 2001
Research on the molecular mechanisms involved in the formation of new vessels from preexisting vasculature (angiogenesis) has increased exponentially in recent years due to its critical role in a number of physiopathological processes such as rheumatoid arthritis, psoriasis, and cancer. Much of the understanding of these in vitro events relies on endothelial cell cultures, 1 To whom correspondence should be addressed. Fax: (⫹34) 91/ 372-0493. E-mail: [email protected].
Analytical Biochemistry 296, 143–147 (2001) doi:10.1006/abio.2001.5200 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
Solid-Phase Extraction of Lipid from Saccharomyces cerevisiae Followed by High-Performance Liquid Chromatography Analysis of Coenzyme Q Content Ruth A. Hagerman, Mark J. Anthony, and Richard A. Willis 1 Division of Nutritional Sciences, University of Texas, Austin, Texas 78712 Received April 6, 2001; published online August 6, 2001
Ubiquinone (coenzyme Q; Q) 2 functions in the mitochondria to transport electrons between complexes I and III of the electron transport chain, and to accept electrons from succinate. All eukaryotic organisms that undergo respiration produce Q, although the specific type of Q varies with the organism. Humans produce Q 10, containing a 10-unit isoprenoid side chain, whereas Saccharomyces cerevisiae (baker’s yeast) produces Q 6. The utility of yeast as a model eukaryote is of interest to many laboratories studying coenzyme Q, but no standard assay of yeast Q 6 content has been established. In this paper, a simple method of lipid extraction followed by solid-phase purification and 1
To whom correspondence should be addressed at Division of Nutritional Sciences, Mail Code A2703, University of Texas at Austin, Austin, TX 78712. Fax: (512) 471-7784. 2 Abbreviations used: Q, coenzyme Q. Analytical Biochemistry 296, 141–143 (2001) doi:10.1006/abio.2001.5224 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
141
HPLC analysis is described. This method was developed to remove the majority of contaminants such as ergosterol from lipid extracts, thus allowing a larger quantity of Q 6 to be analyzed by HPLC. In addition, this method reduces HPLC run times from 70 to 15 min, leading to significant savings of time and solvents. Data generated using this method are presented. Several methods of isolating Q 6 from yeast have been reported. Crane and Barr (1) suggested methanolic saponification to remove ergosterol from acetone extracts of yeast, but reported that yields of Q 6 were much reduced after saponification. Santos-Ocana et al. (2) improved the saponification procedure, but the method required large volumes of solvent. Poon et al. (3) described a sorptive method for removal of ergosterol, which required a second lipid extraction with resultant losses of Q 6 recovery and time. Schultz and Clarke (4) extracted Q 6 from yeast using methanol: petroleum ether. In this method, the yeast cells were dried for 24 h at 80°C, lysed, and extracted. This method was modified slightly by the same laboratory the following year (5). Modifications included drying the cells for 48 h at 37°C, and the use of Q 9 as an internal standard to correct for lipid losses during extraction. However, neither method removes ergosterol from the samples before analysis. We modified the methods of Schultz and Clarke (4) and Jonassen and Clarke (5) as follows. Yeast were grown for 24 h in treatment media and cell pellets were dried under a stream of nitrogen for 2 h at 56°C. Pellet weights obtained from drying under nitrogen were compared to drying for 48 h at 37°C (5) and found to be essentially identical (1.41 and 1.48 mg, respectively). Dry pellets were resuspended in 0.5 ml water, glass beads (0.5 mm) and 1 g of Q 7 (Sigma; from a stock solution of 20 g/ml ethanol) were added, and the suspension was vortexed for 2 min. Ten milliliters of methanol:petroleum ether (6:4) was added, the mixture was vortexed briefly, and 1 ml of water was added to achieve proper phase separation. In order to achieve clear separation of phases, the proportions must be at least 1:6:4 (water:methanol:petroleum ether, v/v), although more water can be added (we used 1.5:6:4). Thus, cell pellets of any weight suspended in a convenient volume of water may be extracted using these proportions. Phases were separated by centrifugation (1000g) and the organic phase (top layer) was reserved. The pellet and lower phase were reextracted once with 4 ml of petroleum ether. Crane and Barr (1) suggested that the lipid extraction should be repeated sequentially to verify that the maximum amount of Q 6 is obtained. In our hands, a representative sequential extraction yielded 164.1 g Q 6/g dry weight of cells in the first extraction, followed by 15.8 g/g in the second, and 1.3 g/g in the third. Thus, our method includes only two extractions
142
NOTES & TIPS
FIG. 1. HPLC analysis of lipid extracted from yeast. Lipids were extracted from yeast and analyzed for Q 6 content (A) as described in the text. An additional solid-phase extraction step removed approximately 96% of the ergosterol (B).
of the pellet. Supernatants were pooled, dried under nitrogen, and stored at 4°C. In Fig. 1A, a representative chromatogram of the crude unfiltered extract is presented. Lipid samples were dried completely under nitrogen and resuspended in a known volume of isopropanol. Samples were analyzed on a Beckman 168 HPLC with a diode array detector using System Gold Nouveau software and a Zorbax ODS reversed-phase column. Compounds were separated using an isocratic mobile phase of hexane: methanol (1:9, v/v), at a flow rate of 1.2 ml/min. Both Q 6 and Q 7 were detected at 275 nm. No peak was visible at 290 nm, the characteristic absorbance region of reduced CoQ. The identities of ergosterol, Q 6, and Q 7 were confirmed by mass spectrometry (Department of Chemistry, The University of Texas at Austin). Under these conditions, the ergosterol peak at 5.3 min (and a derivative at 5.8 min, also seen in the
standard purchased from Sigma) often obscured the Q 6 peak, making analysis difficult. There also were four major compounds eluting late in the run (43.6, 50.8, 58.5, and 67.9 min; data not shown) which necessitated a total run length of 70 min. These peaks were collected from the HPLC, dried, and submitted for identification by mass spectrometry (Department of Chemistry, University of Texas at Austin). A positive identification was not obtained; however, based on the UV absorbance spectra and mass analysis, we believe these compounds are precursors or derivatives of ergosterol. We developed a method using solid-phase extraction to remove the 4 late peaks and ergosterol from the lipid extract before HPLC analysis. In Fig. 1B, a representative chromatogram from a column-treated sample is shown. A 3-cm hand-packed column of 230 – 400 mesh silica gel (Sigma) was prepared in a 5-inch glass pipette and washed with 3 ml of dichloromethane:hexane. The silica was not heat-activated prior to packing the column. The dried lipid extract (5–10 g) was dissolved in 1 ml of dichloromethane:hexane (2.5:1, v/v) and applied to the column. An additional 1 ml of dichloromethane:hexane was added and the eluate collected in 0.5-ml fractions. As shown in Table 1, the majority of the two largest peaks eluted in the first 2 ml. Peaks III and IV also elute in these fractions (data not shown). The next 2 ml of dichloromethane:hexane contained the majority of the Q 6 and Q 7. Approximately 96% of the ergosterol remained on the column and can be eluted with an additional 3 ml of 100% dichloromethane. Thus, for Q 6 analysis, fractions 5 through 8 can be reserved for HPLC analysis and the remainder discarded. HPLC analysis is as described above but run times are cut from 70 to 15 min. Traces of contaminants may remain in the sample after the solid-phase extraction. When run times for Q 6 analysis are reduced to 15 min, a routine 70-min flush of the
TABLE 1
Elution Profile of Solid-Phase Extraction Column a Fraction b (0.5 ml)
Peak I
Peak II
CoQ 6
CoQ 7
Ergosterol
1 2 3 4 5 6 7 8 9 10
306962 1953593 374433 44454 — — — — — —
66073 315246 74462 5354 — — — — — —
— — — 42142 73842 91900 53300 38089 — —
— — — 11577 20873 23056 9993 3579 — —
— — — — — — — — — 5290344
Data are area under the curve from a 5 g sample of lipid. Fractions 1 to 9 are in units of 0.5 ml of dichloromethane:hexane (2.5:1, v/v). Fraction 10 is a wash of 3 ml of dichloromethane. a b
143
NOTES & TIPS
yeast. In addition, we found that cell drying times could be reduced from 2 days to 2 h. Using this method, we have shown that yeast grown on ␥-linolenic acid for 24 h produce less Q 6 than yeast grown on the unsaturated fatty acids, oleic, elaidic, or linoleic, although cells grew to the same final concentration in all media. Acknowledgments. The authors thank Dr. Ann E. Hagerman for critical reading of this manuscript. This research was supported in part by the Karl Folkers Foundation.
REFERENCES FIG. 2. Yeast grown on different fatty acids produce different amounts of Q 6. Yeast were grown for 24 h in the indicated fatty acids and analyzed for Q 6 content as described in the text. Data are expressed as microgram Q 6 per gram dry weight of cells (mean ⫾ SD, n ⫽ 3 to 5).
system with a blank sample is included approximately every 10 runs to remove any trace compounds which may have accumulated on the column. Removal of the majority of the ergosterol from the chromatograms improved quantitation of Q 6 significantly. Some analyte was lost in the solid-phase extraction step, but inclusion of the internal standard (Q 7) provided a convenient method for correcting that loss. Using this method, approximately 30% of the internal standard was lost, compared to typical losses of 2 to 12% in crude extractions. We have used this method successfully to analyze the Q 6 content of yeast grown on different fatty acids. The yeast strain SEY6210 was grown at 30°C while shaking in defined yeast medium (6% yeast nitrogen base, 0.5% yeast extract, 1% tergitol NP-40, and amino acids) containing 1 mM fatty acid (oleic, elaidic, linoleic, or ␥-linolenic). Yeast were harvested at 24 h and Q 6 analyzed as described above. Figure 2 shows that yeast grown on the -9 fatty acids oleic (cis 18:1) or elaidic (trans 18:1) produced the most Q 6 (15.9 ⫾ 2.5 and 18.3 ⫾ 1.6 g/g dry weight of cells, respectively). Slightly less Q 6 was produced after growth on linoleic (cis, cis 18:2, -6; 12.2 ⫾ 2.5 g/g dry weight) and much less after growth on ␥-linolenic (all cis 18:3, -6; 7.3 ⫾ 0.7 g/g dry weight). To compare growth rates, absorbance at 600 nm was monitored. At 24 h, all carbon sources allowed for the growth of cells to the same concentration, although the cells grew at different rates (data not shown). Thus, the differences in cellular Q 6 content were not reflected in cell growth. In conclusion, solid-phase extraction of lipid from yeast yields a fraction containing only 4% of the initial ergosterol. The additional major lipid contaminants are also no longer present. The extracted samples yield cleaner chromatograms and require run times of only 15 min, resulting in significant savings of time and volume of solvent required for analysis of Q 6 from
1. Crane, F. L., and Barr, R. (1971) Determination of ubiquinones. Methods Enzymol. 18, 137–165. 2. Santos-Ocana, C., Cordoba, F., Crane, F. L., Clarke, C. F., and Navas, P. (1998) Coenzyme Q 6 and iron reduction are responsible for the extracellular ascorbate stabilization at the plasma membrane of Saccharomyces cerevisiae. J. Biol. Chem. 273, 8099 – 8105. 3. Poon, W. W., Marbois, B. N., Faull, K. F., and Clarke, C. F. (1995) 3-Hexaprenyl-4-hydroxybenzoic acid forms a predominant intermediate pool in ubiquinone biosynthesis in Saccharomyces cerevisiae. Arch. Biochem. Biophys. 320, 305–314. 4. Schultz, J. R., and Clarke, C. F. (1999) Characterization of Saccharomyces cerevisiae ubiquinone-deficient mutants. Biofactors 9, 121–129. 5. Jonassen, T., and Clarke, C. F. (2000) Isolation and functional expression of human COQ3, a gene encoding a methyltransferase required for ubiquinone biosynthesis. J. Biol. Chem. 275, 12381– 12387.
High Transfection Efficiency of Human Umbilical Vein Endothelial Cells Using an Optimized Calcium Phosphate Method Inmaculada Segura, Manuel A. Gonza´lez, Antonio Serrano, Jose´ Luis Abad, Antonio Bernad, and Hans H. Riese 1 Department of Immunology and Oncology, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas/Universidad Auto´noma de Madrid, Campus de Cantoblanco, E-28049 Madrid, Spain Received April 12, 2001; published online August 3, 2001
Research on the molecular mechanisms involved in the formation of new vessels from preexisting vasculature (angiogenesis) has increased exponentially in recent years due to its critical role in a number of physiopathological processes such as rheumatoid arthritis, psoriasis, and cancer. Much of the understanding of these in vitro events relies on endothelial cell cultures, 1 To whom correspondence should be addressed. Fax: (⫹34) 91/ 372-0493. E-mail: [email protected].
Analytical Biochemistry 296, 143–147 (2001) doi:10.1006/abio.2001.5200 0003-2697/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.