Regulation of Intracellular Ceramide Content in B16 Melanoma Cells

Abstract

We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996)J. Biol. Chem.271, 12655–12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAcα2,3Galβ1,4Glcβ1,1ceramide was significantly increased. Interestingly, UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence ofd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess. We previously reported that ceramide released from glycosphingolipids (GSLs) by endoglycoceramidase was directly metabolized to GSLs, and thus the content of GSLs was constantly maintained in B16 melanoma cells (Ito, M., and Komori, H. (1996)J. Biol. Chem.271, 12655–12660). In this study, the metabolism of ceramide released from sphingomyelin (SM) by bacterial sphingomyelinase (SMase) was examined using B16 cells and their GSL-deficient mutant counterpart GM95 cells. Treatment of B16 melanoma cells with bacterial SMase effectively hydrolyzed SM on the plasma membrane. Under these conditions, NeuAcα2,3Galβ1,4Glcβ1,1ceramide was significantly increased. Interestingly, UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1) activity and GSL synthesis, but not SM synthesis or sphingosine generation, were found to be up-regulated by SMase treatment. The up-regulation of GSL synthesis seemed to occur at both the transcriptional and post-translational steps of GlcT-1 synthesis. Accumulation of ceramide by bacterial SMase was much higher in GM95 cells than in the parental cells. When the enzyme was removed from the culture medium, the intracellular ceramide level in B16 cells, but not that in the mutant cells, normalized. No rapid restoration of SM in either of the cell lines was observed after removal of the enzyme. SMase treatment strongly inhibited DNA synthesis in GM95 cells but not that in B16 cells. In the presence ofd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, an inhibitor of GlcT-1, SMase treatment markedly increased the ceramide content and thus inhibited DNA synthesis in B16 cells. Our study provides the first evidence that GlcT-1 functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess. Glycosphingolipids (GSLs) 1The abbreviations used are:GSL, glycosphingolipid; C6-ceramide, N-hexanoylsphingosine; CMH, ceramide monohexoside; GlcT-1, UDP-glucose:ceramide glucosyltransferase-1; GM3, NeuAcα2,3Galβ1,4Glcβ1,1ceramide; MEM, minimum essential medium; PBS, phosphate-buffered saline; PDMP, d-threo-1-phenyl-2-decanoylamino3-morpholino-1-propanol; SM, sphingomyelin; SMase, sphingomyelinase; TLC, thin layer chromatography; FBS, fetal bovine serum. and sphingomyelin (SM) are characteristic components of vertebrate plasma membranes and have the same hydrophobic residue, ceramide, which consists of a sphingosine and a fatty acid. GSLs have been defined as tumor antigens, receptors for microbes and their toxins, and possible modulators of cell proliferation, differentiation, and cell-cell interactions (1Hakomori S. Annu. Rev. Biochem. 1981; 50: 733-764Crossref PubMed Scopus (1474) Google Scholar, 2Karlsson K.-A. Annu. Rev. Biochem. 1989; 58: 309-350Crossref PubMed Scopus (619) Google Scholar, 3Iwabuchi K. Yamamura S. Prinetti A. Handa K. Hakomori S. J. Biol. Chem. 1998; 273: 9130-9138Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Recently, ceramide has emerged as a novel second messenger for intracellular signaling pathways responding to various cytokines and stress (4Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar). Several lines of evidence indicate that a signaling ceramide is produced from SM by the action of endogenous neutral (5Okazaki T. Bell R.M. Hannun Y.A. J. Biol. Chem. 1989; 264: 19076-19080Abstract Full Text PDF PubMed Google Scholar) and acid sphingomyelinase (SMase; EC3.1.4.12) (6Schwandner R. Wiegmann K. Bernardo K. Kreder D. Kronke M. J. Biol. Chem. 1998; 273: 5916-5922Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), or by de novo synthesis (7Bose B. Verheij M. Haimovitz-Friedman A. Scotto K. Fuks Z. Kolesnick R. Cell. 1995; 82: 405-414Abstract Full Text PDF PubMed Scopus (784) Google Scholar). However, little is known about the mechanism of regulation of intracellular level of ceramide, which should be strictly regulated within cells. Endoglycoceramidase (EC3.2.1.123) is a GSL-specific enzyme fromRhodococcus sp. that hydrolyzes the glycosidic linkage of ceramide and sugar chains of various GSLs (8Ito M. Yamagata T. J. Biol. Chem. 1986; 261: 14278-14282Abstract Full Text PDF PubMed Google Scholar). The cell surface GSLs of various erythrocytes (9Ito M. Ikegami Y. Tai T. Yamagata T. Eur. J. Biochem. 1993; 218: 637-643Crossref PubMed Scopus (17) Google Scholar) and cultured mammalian cells (10Ji L. Ito M. Zhang G. Yamagata T. Glycobiology. 1995; 5: 343-350Crossref PubMed Scopus (16) Google Scholar) were hydrolyzed by the purified rhodococcal endoglycoceramidase (11Ito M. Yamagata T. J. Biol. Chem. 1989; 264: 9510-9519Abstract Full Text PDF PubMed Google Scholar) with the assistance of its protein activator (12Ito M. Ikegami Y. Yamagata T. J. Biol. Chem. 1991; 266: 7919-7926Abstract Full Text PDF PubMed Google Scholar). We found that treatment of B16 melanoma cells with a microbial endoglycoceramidase activated GSL synthesis via transient up-regulation of UDP-glucose:ceramide glucosyltransferase-1 (GlcT-1, glucosylceramide synthase; EC2.4.1.80) (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). As a result, cell surface NeuAcα2,3Galβ1,4Glcβ1,1ceramide (GM3), the end product of GSL synthesis in B16 cells, was restored quickly when the enzyme was removed from the culture medium (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). In this study, we examined the effects of bacterial SMase on the metabolism of GSLs and SM using B16 cells and their GSL-deficient mutant counterpart GM95 cells, which lack GlcT-1 (14Ichikawa S. Nakajo N. Sakiyama H. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2703-2707Crossref PubMed Scopus (142) Google Scholar). Although GSLs were quickly restored after endoglycoceramidase treatment, restoration of SM was not observed after treatment with bacterial SMase in B16 melanoma cells. Interestingly, ceramides generated from not only GSLs but also SM by the microbial enzymes were primarily glucosylated by GlcT-1, metabolized to GM3, and then transported to the plasma membrane. Ceramide was accumulated during SMase treatment in GM95 or B16 cells in the presence ofd-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP) (15Radin N.S. Shayman J.A. Inokuchi J. Adv. Lipid Res. 1993; 26: 183-213PubMed Google Scholar) or N-butyldeoxynojirimycin (16Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar), potent inhibitors of GlcT-1. These results suggest a biological role of GlcT-1 in the regulation of intracellular ceramide content. Because excess generation of ceramide is toxic to cells, GlcT-1 seems to function for expulsion of ceramide from the cell. This regulation, regarded as a putative detoxification mechanism, may function as a defense against an unexpected increase of ceramide, which could be caused by various forms of stress, e.g. infections with pathogenic microorganisms that produce SMase or endoglycoceramidase. This paper also indicates the biological role of ceramide as a modulator of the overall synthesis of GSLs by regulating GlcT-1 at both the transcriptional and post-translational levels. Monoclonal antibody M2590 and fluorescein isothiocyanate-conjugated goat anti-mouse IgM were purchased from Cosmo Bio Co., SMases from Staphylococcus aureus andBacillus cereus were obtained from Sigma and Funakoshi Co., respectively. d-Threo-PDMP andN-hexanoylsphingosine (C6-ceramide) were purchased from Matreya, and thin layer chromatography (TLC) plates (Silica Gel 60) were from Merck. 14C-Labeled Gal, serine, choline, and [32P]ATP were obtained from DuPont NEN, and [3H]thymidine, [14C]palmitic acid, and [14C]UDP-glucose were from American Radiolabeled Chemicals. sn-1,2-Diacylglycerol kinase was kindly provided by Dr. T. Okazaki (Kyoto University, Kyoto, Japan). All other chemicals were of the highest grade available. All experiments using B16 melanoma and the GlcT-1-deficient mutant GM95 cells (14Ichikawa S. Nakajo N. Sakiyama H. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2703-2707Crossref PubMed Scopus (142) Google Scholar) were conducted at 37 °C in minimum essential medium (MEM) supplemented with 5% fetal bovine serum (FBS) in a humidified 95% air, 5% CO2incubator. SMase from S. aureus was used for all experiments except that shown in Fig. 2, in which the enzyme from B. cereus was used. Cells were incubated in 200 μl of MEM supplemented with 5% FBS containing 1 μCi of [14C]Gal, [14C]serine, or [14C]choline for the times indicated. Cells (1 × 105) were harvested by centrifugation (2000 rpm for 5 min) and washed twice with phosphate-buffered saline (PBS). Sphingolipids were extracted with 750 μl of i-propanol/hexane/water (55:35:10, v/v) in a sonicator for 20 min. After centrifugation at 13,000 rpm for 5 min, the supernatants obtained were dried under N2 gas, dissolved in 25 μl of chloroform/methanol (2:1, v/v), and applied to TLC plates, which were developed with chloroform/methanol/0.02% CaCl2(5:4:1, v/v) for GSLs and chloroform/methanol/acetic acid/H2O (50:30:8:5, v/v) for SM. Radiolabeled sphingolipids separated on TLC plates were analyzed and quantified using a BAS1000 imaging analyzer (Fuji Film). Identification of C6-ceramide GSLs was done by the method described by Komori and Ito (17Komori H. Ito M. FEBS Lett. 1995; 374: 299-302Crossref PubMed Scopus (14) Google Scholar). The radiolabeled total lipid extracts from cells were spotted onto TLC plates (10 × 10 cm). The first chromatographic run was performed with chloroform/methanol/formic acid/H2O (65:25:8.9:1.1, v/v). The second run was performed with chloroform/methanol/5 n NH4OH (50:40:10, v/v) perpendicular to the original direction. Then the third run was performed with diethylether in the opposite direction to the second run to separate ceramides from other neutral lipids. Spots on the TLC plates were identified using authentic standards (18Yokoyama K. Nojiri H. Suzuki M. Setaka M. Suzuki A. Nojima S. FEBS Lett. 1995; 368: 477-480Crossref PubMed Scopus (22) Google Scholar). Total lipids, extracted from 5–7 × 106 cells with a mixture ofi-propanol/hexane/water (55:35:10, v/v), were applied to TLC plates, which were then developed with chloroform/methanol/acetic acid/H2O (50:30:8:5, v/v). SM was visualized with Coomasie Brilliant Blue (19Nakamura K. Handa S. Anal. Biochem. 1984; 142: 406-410Crossref PubMed Scopus (182) Google Scholar) and quantified using a Shimadzu CS-9300 chromatoscanner with reflection mode set at 580 nm. Ceramide was extracted from cells (2 × 106) with 3 ml of chloroform/methanol (1:2, v/v), and 0.8 ml of water was added and mixed well. The organic and aqueous phases were subsequently separated by addition of 1 ml of chloroform and 1 ml of water followed by vigorous shaking and centrifuged at 3000 rpm. The organic phase was carefully removed and transferred to a new tube, and the samples were dried under N2 gas. The of ceramide was kinase as described R. Hannun Y.A. J. PubMed Scopus Google Scholar). Ceramide and acid were separated by TLC using acid/H2O v/v) as a B16 cells (1 × 106) were incubated on for with μl of antibody M2590 to Y. A. M. T. S. M. J. Biol. Chem. Full Text PDF PubMed Google Scholar). After twice with PBS, B16 cells were with μl of antibody isothiocyanate-conjugated goat anti-mouse on for and then analyzed by Cells (2 × were with 20 of SMase in 200 μl of MEM supplemented with 5% FBS in plates at 37 °C for After 10 μl of containing μCi of was added to the After at 37 °C for cells were using a cell and the of DNA was quantified by GlcT-1 activity was to the method of S. B. S. J. Biol. Chem. Full Text PDF PubMed Google Scholar) with cell cells were washed with 1 ml of and in μl of 10 containing and 5 mixture [14C]UDP-glucose ceramide and cell as in 20 After at °C for μl of chloroform/methanol (2:1, v/v) was added to the and the layer was applied to TLC plates, which were then developed with v/v). The produced was with an imaging analyzer Film). of was mixed with of and 10 of in 1 ml of water to cell cells × 105) were washed with 1 ml of and were in μl of 10 The mixture 10 μl of 1 and cell as in μl of After at °C for min, μl of chloroform/methanol (2:1, v/v) was added to the and the layer was applied to TLC plates, which were then developed with v/v). The produced was with a Shimadzu CS-9300 chromatoscanner The activity by this method to the total activity of and of cell extracts were by the method with bovine serum as the Total was from cells with an of of total were to on containing and onto membranes The of the GlcT-1 was with by the DNA and used as a was at °C for in 5 × containing 5 × DNA S. Sakiyama H. Suzuki G. Hirabayashi Y. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). After the was washed with × containing 1 × containing and × containing at °C for at The membranes were also by the method described but with a for of levels. were quantified using an imaging analyzer Film). C6-ceramide was added to cells (1 × 106) and incubated at 37 °C in a for the times indicated. Cells were harvested by centrifugation (2000 rpm for 5 min) and washed twice with Sphingolipids were extracted with 750 μl ofi-propanol/hexane/water (55:35:10, v/v) in a sonicator for 20 min. After centrifugation at 13,000 rpm for 5 min, the supernatants obtained were dried under N2 gas, dissolved in 25 μl of chloroform/methanol (2:1, v/v), and applied to TLC plates, which were then developed with v/v). The produced were with a Shimadzu CS-9300 chromatoscanner We previously reported that endoglycoceramidase treatment of B16 melanoma cells GSL synthesis (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Because ceramide is the lipid of GSLs and SM, we examined the metabolism of ceramide using bacterial SMase of endoglycoceramidase. The ceramide of SM and GSLs of B16 cells were with [14C]serine, washed with MEM, and then with SMase from S. TLC that SMase treatment markedly the content of to of that in that of increased to of the level 1 the of de novo synthesis of ceramide, the same was conducted but in the presence of an inhibitor for Adv. Lipid Res. 1993; 26: Google Scholar). in the presence of the increase in with the in SM was observed after SMase treatment that the increase in level was not to an increase in de novo synthesis of SMase treatment, ceramide and was increased in B16 cells However, the level of sphingosine was not after SMase treatment not These results strongly suggest that increased was metabolized from ceramide by glycosylation but not from from of The increase of was also by using a that the increased was transported to the cell surface increase in GSL synthesis during SMase treatment was also observed in not only B16 cells but also other cell as shown by the synthesis of in cells, which increased by not than during SMase treatment. The GlcT-1 activity and de novo synthesis of GSLs in B16 cells were examined with or SMase treatment. Interestingly, GlcT-1 activity in the cell increased by by the enzyme treatment, SM activity was not was that GM95 cells GlcT-1 activity not The of GlcT-1 seems to occur in the presence of an increase in GSL synthesis was observed after SMase treatment the other that SMase treatment activated of the GlcT-1 Treatment of B16 cells with C6-ceramide also increased GlcT-1 in a These results indicate that up-regulation of GSL synthesis by SMase treatment at the level of both and of the GlcT-1 and also that ceramide could the overall synthesis of GSLs via regulation of GlcT-1. The content of in B16 cells was by endoglycoceramidase treatment but quickly after the enzyme was removed from the culture medium (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, restoration in SM content was observed after removal of SMase when B16 cells were with [14C]choline GM95 cells also rapid restoration of SM should be that this restoration was by a ceramide and which is to be the of SM synthesis 1984; PubMed Scopus Google Scholar). However, a of SM to in B16 cells after removal of the enzyme and for when the SM was by Coomasie Brilliant Blue This increase in SM seemed to be from de of ceramide and not from the of The ceramide content of B16 cells, which was kinase increased by after SMase treatment for 3 but after removal of the enzyme and to the level after In in GlcT-1-deficient mutant GM95 cells the of ceramide by SMase treatment was much higher than in the parental cells and was maintained after the enzyme was removed from the culture medium Interestingly, SMase treatment of B16 cells in the presence of PDMP, a potent inhibitor of GlcT-1, markedly increased the of ceramide Ceramide generation in B16 cells by bacterial SMase was also times by addition at 200 which is an inhibitor of GlcT-1 (16Platt F.M. Neises G.R. Dwek R.A. Butters T.D. J. Biol. Chem. 1994; 269: 8362-8365Abstract Full Text PDF PubMed Google Scholar). These results suggest that GlcT-1 functions to the excess ceramide generated by bacterial Although ceramide functions as a novel of second messenger (4Hannun Y.A. Science. 1996; 274: 1855-1859Crossref PubMed Scopus (1497) Google Scholar), the excess generation of ceramide be to the cell. As shown in DNA synthesis of GM95 cells by the of was significantly inhibited by bacterial SMase treatment, this enzyme treatment seemed to have on that of B16 cells However, in the presence of PDMP, SMase treatment to be for B16 cells 5 The of of DNA synthesis was with that of of ceramide S. T. Sakiyama H. Hirabayashi Y. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that the treatment of GM95 cells with the bacterial SMase and the cells were from However, in this a much of SMase was used to the The effects of the ceramide, C6-ceramide, on DNA synthesis of B16 and GM95 cells were also with C6-ceramide for 3 strongly DNA synthesis in GM95 cells but not in B16 cells 5 This is with that using SMase 5 When C6-ceramide was added to B16 cell in the presence of [14C]Gal, and were both of which were also released the medium However, glycosylation of the ceramide was not found in of GM95 cells the for of ceramide to GSLs and SM, was added to of B16 and GM95 cells. The was in B16 cells much than in GM95 cells In B16 cells, was to and at the same by min, but the generation of was much than that of In GM95 cells, was to but not to and the a by when of in the cells. These results that the of ceramide in B16 cells was by The for glycosylation of ceramide seems to be to ceramide generated from GSLs (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) and SM on the plasma the de novo synthesis of and SM in B16 cells was when was used as a and for and also In this study has the biological role of GlcT-1 in regulation of the intracellular ceramide and of GlcT-1 results in of ceramide and of DNA synthesis after SMase treatment in B16 melanoma cells. Recently, Zhang B. Hannun Y.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported the of a B. cereus SMase in cells. After the of the and of the SMase, intracellular ceramide content in of However, added B. cereus SMase, a of ceramide not in cells B. Hannun Y.A. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). This the of SM of which is by endogenous SMase and the other by The seems to be for of the and the of these be the other that bacterial SMase biological effects on cells. the SMase the action of in cell T. A. Bell R.M. Hannun Y.A. J. Biol. Chem. Full Text PDF PubMed Google Scholar), and the enzyme from S. aureus of cells G. B. S. 1996; PubMed Scopus Google Scholar). In the present study, treatment of B16 cells with bacterial SMase inhibited DNA synthesis under of or of GlcT-1. These results suggest that the and of SM and their to SMase to cell and Hannun Hannun Y.A. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar) reported that treatment of cells with bacterial SMase in a in SM level and generation of This ceramide level in the cells, but was little restoration of SM content. In cells, in which the activity of SM was found to be higher than that in parental cells, ceramide much and SM. The the of SM for regulation of intracellular ceramide in the We on the other that in B16 melanoma cells the ceramides generated from SM as as GSLs (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar) by microbial enzymes were primarily glucosylated by GlcT-1 and metabolized to This may be to the GlcT-1 and SM in cells, which is defined on the of cells or their and be by other The present study that ceramide generated on the of the plasma by bacterial SMase was but not via the R. G. T. T. Res. 1998; PubMed Scopus Google Scholar), metabolized to GSLs in B16 melanoma cells. Because the of GlcT-1 is on the of the D. A. G. J. Biol. PubMed Scopus Google Scholar), the generated ceramide be to the of the it to the enzyme. Although the of ceramide to the suggest that the of ceramide in M. K. J. Biol. Chem. PubMed Scopus Google Scholar) and T. K. D. Biochem. J. 1998; PubMed Scopus Google Scholar) is pathogenic and microbes produce of which have been identified as and Rev. 1993; PubMed Google Scholar). These indicate that cell surface SM of be to the action of microbial SMase, which may in the of the intracellular ceramide Because the excess generation of ceramide be toxic for the the of ceramide from the cell by glycosylation be regarded as a mechanism of defense against by is to that also produce endoglycoceramidase (13Ito M. Komori H. J. Biol. Chem. 1996; 271: 12655-12660Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). Y. H. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. H. A. A. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) reported that cells of glucosylceramide with the parental cells. The was found to GlcT-1, in a in the level of glucosylceramide and an increase in that of This as as an inhibitor of GlcT-1, the cells to These results that GlcT-1 is in regulation of ceramide which may the of cells to We that GlcT-1, in vertebrate cells, functions to regulate the level of intracellular ceramide by glycosylation of the ceramide when it is present in excess. We Dr. of and Dr. Yamagata of of for this We are also to Dr. Okazaki of for the