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Programmed cell death in the epidermis
TIBS 15 - MARCH 1 9 9 0
The quantitation of PQQ with hydrazine-based reagents is generally difficult because of the special reactivity of PQQ and its proclivity to be partially reduced to its hydroxyquinone form by either the detecting reagent or by redox interactions with other components present. The hydroxyquinone of PQQ does not react with carbonyl reagents, resulting in overall low yields. Reaction of PQQ with hydrazine-based reagents carried out in strong acid also causes problems, since PQQ appears to undergo lactone formation with masking of the carbonyl groups :~. Other reaction difficulties have been noted by Duine and his collaborators in several of their papers and this prompted their search for alternate procedures and derivatives, such as hexanol and aqueous HCI4 which appears to allow improved derivatization of the PQQ with several quinoproteins available in significant amounts. This method may still lack the sensitivity required for the quantitation of smaller amounts of PQQ present in tissues, biological fluids and foods. With such samples and for the monitoring of PQQpeptides during HPLC analysis, a simple, specific and sensitive amplification procedure is needed. The redox-cycling NBT-glycinate method fulfills these requirements and can be very specific for PQQ when proper controls are run to check for the presence of interfering compounds r;. Our results with the glycinate-NBT reagent have been confirmed and extended in Rucker's laboratory ~, where comparisons of PQQ measurements by redox-cycling were made with the quinoprotein-holoenzyme
Programmed cell death in the epidermis In a recent article I Tim Hunt gives a fascinating sketch of some of the processes leading to terminal differentiation of the reticulocyte. Although he comments - correctly - that cells of the eye lens undergo a similar fate, he misses what is one of the largest and most interesting tissues of the body, namely the outer epithelium. Mammalian skin is composed of a layer of connective tissue (dermis) supporting a stratified epithelium (epidermis). The latter is made up of three compartments: an innermost layer of dividing keratinocytes, an intermediate region of differentiated keratinocytes and a superficial layer comprising an orderly array of cornified envelopes embedded in a hydrophobic lipid matrix. It is this last layer which is the true chemical barrier between the body and its environment. Not only does it possess remarkable
reconstitution method of Geiger and G6risch 7. This ground-breaking report from Rucker's laboratory documents, for the first time, the essential nutrient and likely vitamin function of PQQ in mammals. We have applied the NBT-glycinate procedure for the detection of PQQcontaining peptides, separated by HPLC from pronase digests of two putative quinoproteins, dopamine-~hydroxylase (DBH) and diamine oxidase (histaminase) and for detecting PQQ directly in several proteins as well as for noting its absence in others. Our method independently confirms the putative quinoprotein nature of some mammalian proteins, reported by Duine's group. Other laboratories who have attempted to use the hydrazine reagent approach to quinoprotein detection are not convinced about the quinoprotein nature of DBH. We and others have noted recently that quinoproteins can be electroblotted onto nitrocellulose and detected specifically by redox-cycle staining with NBTglycinate. Dr Duine questions the presence of PQQ in choline dehydrogenase reported by Ameyama et al. ~. Evidence supporting its presence was based on holoenzyme reconstitution with putative PQQ removed from the choline dehydrogenase. We have had no direct experience with this enzyme ourselves. Serum albumin, obtained commercially, does contain PQQ as detected in our lab by redox-cycling, but after TCA precipitation, redissolved serum albumin is free of PQQ. Putative PQQ derived from serum albumin has also been reported to result in
apoquinoprotein activation 9 in a holoenzyme reconstitution assay. We trust that our response clarifies some of the issues raised by Dr Duine. We also hope that it will help to stimulate further research in this important emerging field.
mechanical and physical properties which make animal life possible outside the sea, but also it is being continuously renewed from the inside (with a turnover time of about three weeks ~) to compensate for natural wear and tear. Thus - like red cells - the keratinocyte only begins to be useful when it has lost its internal organization. Unlike erythropoeitic tissue, however, mammalian epidermis is readily accessible for study, both in vivo and in vitro, and several groups around the world are now exploring the process of 'programmed suicide' involved. Current evidence suggests that the key may lie in a specialized organelle (originally termed the Odland body, now called the lamellar body) which appears in the upper regions of the viable, differentiated compartment. This seems to be a modified lysosome, containing a battery of hydrolytic enzymes (including phospholipases and proteases :~'4)which are presumably released around the time of death. The signal which triggers this event, however, remains quite unknown.
Does the epidermal lamellar body contain a lipoxygenase? Does the reticulocyte possess an organelle whose function is analogous to the lamellar body? A systematic comparison of terminal differentiation in these two cell types could provide answers to many outstanding questions.
References 1 Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J. and Duine, J. A. (1984) FEBS Lett. 170, 305-309 2 Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E. and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565 3 Gallop, P. M., Henson, E., Paz, M. A., Greenspan, S. L. and Fluckiger, R. (1989) Biochem. Biophys. Res. Commun. 163, 755-763 4 van der Meer, R., Mulder, A. C., Jongejan, J. A. and Duine, J. A. (1989) FEBS Lett. 254, 99-105 5 Paz, M. A., FIL~ckiger, R., Henson, E. and Gallop, P. M. (1989) in PQQ and Quinoproteins, (Jongejan, J. A. and Duine, J. A., eds), pp. 131-143, Kluwer Academic Publishers 6 Killgore, J., Smidt, C. et al. (1989) Science 245, 850-852 7 Geiger, O. and Gorisch, H. (1987) Anal. Biochem. 164,418-423 8 Ameyama, M., Shinagawa, E., Matsushita, K., Takimoto, K., Nakashima, K. and Adachi, O. (1985) Agric. Biol. Chem. 49, 3623-3626 9 Adachi, 0., Shinagawa, E., Matsushita, K., Nakashima, K., Takimoto, K. and Ameyama, M. (1989) in PQQ and Quinoproteins (Jongejan, J. A. and Duine, J. A., eds), pp. 145-147, Kluwer Academic Publishers
PAUL M. GALLOP Laboratory of Human Biochemistry, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
References 1 Hunt, T. (1989) Trends Biochem. Sci. 14, 393-394 2 Bauer, F. W. (1986)in Textbook of Psoriasis (Mier, P. D. and van de Kerkhof, P. C. M., eds), pp. 100-112, Churchill-Livingstone 3 Grayson, S., Johnson-Winegar, A. G., Wintroub, B. V., Isseroff, R. R., Epstein, E. H. and Elias, P. M. (1985) J. Invest. Dermatol. 85, 289-294 4 Freinkel, R. K. and Traczyk, T. N. (1985) J. Invest. Dermatol. 85,295-298
P. D. MIER AND P. C. M. VAN DE KERKHOF Department of Dermatology, University Hospital of Nijmegen, Nijmegen, The Netherlands.
97
The quantitation of PQQ with hydrazine-based reagents is generally difficult because of the special reactivity of PQQ and its proclivity to be partially reduced to its hydroxyquinone form by either the detecting reagent or by redox interactions with other components present. The hydroxyquinone of PQQ does not react with carbonyl reagents, resulting in overall low yields. Reaction of PQQ with hydrazine-based reagents carried out in strong acid also causes problems, since PQQ appears to undergo lactone formation with masking of the carbonyl groups :~. Other reaction difficulties have been noted by Duine and his collaborators in several of their papers and this prompted their search for alternate procedures and derivatives, such as hexanol and aqueous HCI4 which appears to allow improved derivatization of the PQQ with several quinoproteins available in significant amounts. This method may still lack the sensitivity required for the quantitation of smaller amounts of PQQ present in tissues, biological fluids and foods. With such samples and for the monitoring of PQQpeptides during HPLC analysis, a simple, specific and sensitive amplification procedure is needed. The redox-cycling NBT-glycinate method fulfills these requirements and can be very specific for PQQ when proper controls are run to check for the presence of interfering compounds r;. Our results with the glycinate-NBT reagent have been confirmed and extended in Rucker's laboratory ~, where comparisons of PQQ measurements by redox-cycling were made with the quinoprotein-holoenzyme
Programmed cell death in the epidermis In a recent article I Tim Hunt gives a fascinating sketch of some of the processes leading to terminal differentiation of the reticulocyte. Although he comments - correctly - that cells of the eye lens undergo a similar fate, he misses what is one of the largest and most interesting tissues of the body, namely the outer epithelium. Mammalian skin is composed of a layer of connective tissue (dermis) supporting a stratified epithelium (epidermis). The latter is made up of three compartments: an innermost layer of dividing keratinocytes, an intermediate region of differentiated keratinocytes and a superficial layer comprising an orderly array of cornified envelopes embedded in a hydrophobic lipid matrix. It is this last layer which is the true chemical barrier between the body and its environment. Not only does it possess remarkable
reconstitution method of Geiger and G6risch 7. This ground-breaking report from Rucker's laboratory documents, for the first time, the essential nutrient and likely vitamin function of PQQ in mammals. We have applied the NBT-glycinate procedure for the detection of PQQcontaining peptides, separated by HPLC from pronase digests of two putative quinoproteins, dopamine-~hydroxylase (DBH) and diamine oxidase (histaminase) and for detecting PQQ directly in several proteins as well as for noting its absence in others. Our method independently confirms the putative quinoprotein nature of some mammalian proteins, reported by Duine's group. Other laboratories who have attempted to use the hydrazine reagent approach to quinoprotein detection are not convinced about the quinoprotein nature of DBH. We and others have noted recently that quinoproteins can be electroblotted onto nitrocellulose and detected specifically by redox-cycle staining with NBTglycinate. Dr Duine questions the presence of PQQ in choline dehydrogenase reported by Ameyama et al. ~. Evidence supporting its presence was based on holoenzyme reconstitution with putative PQQ removed from the choline dehydrogenase. We have had no direct experience with this enzyme ourselves. Serum albumin, obtained commercially, does contain PQQ as detected in our lab by redox-cycling, but after TCA precipitation, redissolved serum albumin is free of PQQ. Putative PQQ derived from serum albumin has also been reported to result in
apoquinoprotein activation 9 in a holoenzyme reconstitution assay. We trust that our response clarifies some of the issues raised by Dr Duine. We also hope that it will help to stimulate further research in this important emerging field.
mechanical and physical properties which make animal life possible outside the sea, but also it is being continuously renewed from the inside (with a turnover time of about three weeks ~) to compensate for natural wear and tear. Thus - like red cells - the keratinocyte only begins to be useful when it has lost its internal organization. Unlike erythropoeitic tissue, however, mammalian epidermis is readily accessible for study, both in vivo and in vitro, and several groups around the world are now exploring the process of 'programmed suicide' involved. Current evidence suggests that the key may lie in a specialized organelle (originally termed the Odland body, now called the lamellar body) which appears in the upper regions of the viable, differentiated compartment. This seems to be a modified lysosome, containing a battery of hydrolytic enzymes (including phospholipases and proteases :~'4)which are presumably released around the time of death. The signal which triggers this event, however, remains quite unknown.
Does the epidermal lamellar body contain a lipoxygenase? Does the reticulocyte possess an organelle whose function is analogous to the lamellar body? A systematic comparison of terminal differentiation in these two cell types could provide answers to many outstanding questions.
References 1 Lobenstein-Verbeek, C. L., Jongejan, J. A., Frank, J. and Duine, J. A. (1984) FEBS Lett. 170, 305-309 2 Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E. and Adachi, O. (1984) Agric. Biol. Chem. 48, 561-565 3 Gallop, P. M., Henson, E., Paz, M. A., Greenspan, S. L. and Fluckiger, R. (1989) Biochem. Biophys. Res. Commun. 163, 755-763 4 van der Meer, R., Mulder, A. C., Jongejan, J. A. and Duine, J. A. (1989) FEBS Lett. 254, 99-105 5 Paz, M. A., FIL~ckiger, R., Henson, E. and Gallop, P. M. (1989) in PQQ and Quinoproteins, (Jongejan, J. A. and Duine, J. A., eds), pp. 131-143, Kluwer Academic Publishers 6 Killgore, J., Smidt, C. et al. (1989) Science 245, 850-852 7 Geiger, O. and Gorisch, H. (1987) Anal. Biochem. 164,418-423 8 Ameyama, M., Shinagawa, E., Matsushita, K., Takimoto, K., Nakashima, K. and Adachi, O. (1985) Agric. Biol. Chem. 49, 3623-3626 9 Adachi, 0., Shinagawa, E., Matsushita, K., Nakashima, K., Takimoto, K. and Ameyama, M. (1989) in PQQ and Quinoproteins (Jongejan, J. A. and Duine, J. A., eds), pp. 145-147, Kluwer Academic Publishers
PAUL M. GALLOP Laboratory of Human Biochemistry, Children's Hospital, 300 Longwood Avenue, Boston, MA 02115, USA.
References 1 Hunt, T. (1989) Trends Biochem. Sci. 14, 393-394 2 Bauer, F. W. (1986)in Textbook of Psoriasis (Mier, P. D. and van de Kerkhof, P. C. M., eds), pp. 100-112, Churchill-Livingstone 3 Grayson, S., Johnson-Winegar, A. G., Wintroub, B. V., Isseroff, R. R., Epstein, E. H. and Elias, P. M. (1985) J. Invest. Dermatol. 85, 289-294 4 Freinkel, R. K. and Traczyk, T. N. (1985) J. Invest. Dermatol. 85,295-298
P. D. MIER AND P. C. M. VAN DE KERKHOF Department of Dermatology, University Hospital of Nijmegen, Nijmegen, The Netherlands.
97