John Leonora

John Leonora (January 30, 1928 - February 17, 2006), is notable for his research into the critical role of hypothalamic "factors" for indirectly controlling the metabolism of such avascular tissues as the dental enamel, the dentin, and the Islands of Langerhans (pancreas). His contributions have given a radically new perspective to the systemic role of the brain in both health and disease. Most of his research was carried out during his 45 years as a faculty member at Loma Linda University and instructor at the university's predecessor, the College of Medical Evangelists.

Biographical Information

John Leonora, the son of Joseph Leonora (March 14, 1883 - November 20, 1942) and Carmela Folise Leonora (April 29, 1888-June 20, 1965), was born in Milwaukee, Wisconsin, soon after his parents arrived as immigrants from Sicily. They had left their eldest daughter in the Sicilian province of Enna where she had already grown to adulthood, married and had children. Their second daughter had died of influenza in Sicily near the end of World War I.

After Joseph Leonora died in 1942, Carmela Leonora found employment as a hand stitcher in an Italian shoe factory. John became an errand boy for various businesses throughout Milwaukee. At the same time he excelled in school and became a proficient pianist, accompanying and playing in jazz combos. He was an active member of the Italian club at Lincoln High School. Because of excellent grades he was awarded a one-year scholarship at the University of Wisconsin-Milwaukee.

Upon receiving his B.S. degree at the University of Wisconsin-Madison, John took an interim position as a laboratory assistant in chemistry and obtained teaching credentials at what is now Andrews University, Berrien Springs, Michigan. There he met Johanna Mae Zwemer, who was working toward a secretarial degree. They later married in 1952. Meanwhile Leonora had returned to Madison and the University of Wisconsin. There he and his wife lived while he completed his Ph.D., obtained a federal fellowship, and embarked on his career as an endocrinologist.

For more than four decades, Dr. Leonora was active in his chosen field of medical physiology. He not only mentored numerous graduate students, but provided leadership for research conducted in his own laboratory. In his research he demonstrated surgical skills on animals as diverse as a 200 pound pig or a mouse weighing only a few ounces. He also delighted in teaching. His lectures were remembered by his students for their clarity and quiet humor.

John was profoundly dedicated to his family—his wife, Johanna, daughters Carmela and Andrea, and three grandchildren. In his leisure time he delighted in the quietude of his country home. He spent hours working in the yard and cooking in the kitchen. He was famous for his homemade pizza, pasta sauce, and biscotti.

Dr. Leonora’s compassion, his career and his contributions will never be forgotten. He literally opened the portal to a new, dynamic, interactive view of the brain’s function in disease, in health, and in life itself.

Education

1949: B.S. in Zoology and Chemistry, University of Wisconsin, Madison, WI

1954: M.S. in Zoology, University of Wisconsin, Madison, WI

1957: Ph.D. in Endocrinology, University of Wisconsin, Madison, WI

Title of Dissertation: Separation and Purification of Follicle Stimulating and Luteinizing Hormone from Ovine Pituitary Glands

1957-59: NIH Postdoctoral Fellow, University of Wisconsin, Madison, WI

Academic Appointments

1952-1957: Research Assistant, Department of Zoology and Endocrinology, University of Wisconsin, Madison, WI

1959-1963: Instructor, Department of Medicine, College of Medical Evangelists, Los Angeles, CA

1963-1968: Assistant Professor, Department of Physiology, Loma Linda University, Loma Linda, CA

1968-1970: Associate Professor, Department of Physiology, Loma Linda University

1970-2002: Professor and Co-Chairman, Department of Physiology and Pharmacology, Loma Linda University

Academic Career

Loma Linda University

Lectures

School of Medicine:

• Fluid and Electrolyte Homeostasis
• Endocrine Physiology

School of Dentistry:

• Systemic Factors in Dental Health
• Calcium Homeostasis
• Hormonal Factors in Dental Health

Graduate School:

• Hormonal Control of Nutrient Homeostasis

Committee Memberships

• Member, Executive Committee, School of Medicine
• Member, Graduate School Council
• Member, Scholarship Committee, Graduate School

Invited Lectures and Symposia

• Systemic effects of sugar on the hypothalamic-parotid gland endocrine axis. Alumni Postgraduate Convention, Loma Linda University, February 1974.

• Systemic effects of sugar on the hypothalamic-parotid gland endocrine axis. Dietetics Convention, School of Health, Loma Linda University, 1974.

• Role of systemic resistance as mediated by hormone action in controlling the incidence of dental caries. Alumni Postgraduate Convention, School of Medicine, Loma Linda University, March 1975.

• Role of trace elements in glucose metabolism, Southern California Nutrition Society, Fullerton, CA, March 1978.

• Physiological observations of a new parotid hormone. The Endocrine Society, Washington, D.C., June 1980.

• Role of hormones in cariostasis. Continuing education lecture, School of Dentistry, Loma Linda University, April 1981.

• New concepts in dental caries. Second Annual Nutrition Symposium, Loma Linda University, May 1983.

• Physiologic significance of the hypothalamic-parotid gland endocrine axis as it relates to dental health. Weimar Institute, Weimar, CA, May 1988.

Academic Recognition

Professional Membership

1959-2006: The Endocrine Society

Academic Awards

1976: Walter E. Macpherson Society, Research Award

1980: Walter E. Macpherson Society, Basic Science Teacher Award

1980: Sigma Xi Research Merit Award

1988: Walter E. Macpherson Society Basic Science Teacher Award

1988: Sigma Xi Research Merit Award

2005: Honorary Omicron Kappa Upsilon Award by the Chi Chi Chapter, Loma Linda University.

Research Grant Awards

In 40 years of active investigation, John Leonora received over one million dollars in research grant awards from such entities as the National Institutes of Health, the Walter E. Macpherson Society, the Don Baxter Foundation, the Pediatric Research Endowment Fund and the Basic Science Research Fund.

Research Contributions

Gonadotropic Hormones. Based on his doctoral studies and subsequent research fellowship, John Leonora initially intended to devote his career to the investigation of gonadotropic hormones.

Dentinal Fluid Transport. One day he received a telephone call from Dr. Ralph R. Steinman, a dental colleague, who had been studying the flow of dentinal fluid in rats from the odontoblasts in the dental pulp through the dentin using an intraperitoneal injection of the fluorescent dye, acriflavine hydrochloride. He found that in the teeth of rats fed a cariogenic diet the flow of dentinal fluid was markedly reduced. He wondered if some systemic mechanism was involved in this impairment and so decided to contact Dr. John Leonora, as an endocrinologist.

Hypothalamus. This was the beginning of a decades-long collaborative journey. John Leonora first suggested that none of the recognized hormones were plausible candidates for regulating dentinal flow transport (DFT), but that the hypothalamus might well be an alternative hormonal source. Quickly they found that infusing rats with a crude extract from rabbit hypothalami triggered increased DFT activity. This, however, raised the question whether the hypothalamic factor had a direct effect on the teeth or rather had an indirect effect characteristic of other hypothalamic hormones.

Parotid Hormone. Assuming that the hypothalamic factor was mediated through one of the major salivary glands, they found that this factor was biologically active when administered to rats with intact parotid glands but was wholly ineffective in rats that had the parotid glands removed. There was no involvement of the other salivary glands. They concluded that the direct regulation of DFT was therefore secreted by the parotid glands and this endocrine function was controlled by the hypothalamus.

Purification of the Hormones. The next step was to isolate the purified parotid hormone from porcine glands, determine its amino acid structure and then confirm its stimulation of DFT. Then the hypothalamic parotid hormone releasing factor was partially purified but not completed because of the rejection of a research grant application. While the research was thus diverted it persisted in other directions.

Antibody to the Parotid Hormone. First, an antibody to the porcine parotid hormone was produced, isolated and used for a radio-immunoassay of the parotid hormone. Later an ELISA method (enzyme linked immunoadsorbent assay) was developed for measuring the parotid hormone titer in biological fluids.

Suppressive Effect of Sucrose. Then the research focused on the mechanism by which dietary sucrose suppressed the DFT. Initial studies suggested that the sucrose suppressed the secretion of the parotid hormone. Further studies, however, showed that the sucrose effect occurred indirectly by inhibiting secretion of the hypothalamic parotid hormone releasing factor. Then it was found that the sucrose effect could be effectively reversed by the infusion of the compound carbamyl phosphate through the internal carotid artery. This confirmed that the site of action was within the central nervous system—namely the hypothalamus.

Effect of Carbamyl Phosphate. Not surprisingly, intact rats fed carbamyl phosphate along with a cariogenic diet showed a highly significant reduction in caries. Carbamyl phosphate, however, was ineffective in parotidectomized rats. Therefore, an intact hypothalamic-parotid gland endocrine axis was found to be necessary for the effectiveness of carbamyl phosphate.

Neural Stimuli of the Hypothalamus. The next question was, what physiological factors normally stimulate the secretion of the parotid hormone? Feeding pigs a standard pig chow proved to effectively stimulate parotid hormone secretion along with the copious secretion of saliva. Surprisingly, pigs fed nonnutritive substances and exposed to auditory, olfactory and visual cues salivated profusely but these cues did not change the fasting level of the parotid hormone. This demonstrated that, although the endocrine and exocrine functions of the parotid glands occur concurrently, they must be controlled by different mechanisms. The nutritive composition thus proved to be critical for the secretion of the parotid hormone. Apparently nutritive substrates activate neural stimuli going to the hypothalamus, which in turn activates the hypothalamic-parotid gland endocrine axis that then stimulates the dentinal fluid transport mechanism.

Formation of Dentin. At this critical juncture a group of investigators at the University of Oulu Dental School, Oulu, Finland (Tjaderhane, L. et al, (1994) discovered that the parotid hormone not only regulates the flow of dentinal fluid but also is involved in the formation of dentin—dentinogenesis. They also found that a high sucrose diet in young rats suppressed primary dentinogenesis. The injection of carbamyl phosphate significantly reversed the effect of sucrose—but only in intact rats. Furthermore, carbamyl phosphate prevented the atrophy of the parotid glands associated with the ingestion of sucrose.

Synthesis of the Parotid Hormone. Subsequent research successfully cloned the porcine cDNAs that code for the parotid hormone gene. The amino acids encoded by these cDNAs agreed with the amino acid sequence of the hormone isolated from the porcine parotid glands. In addition, a commercially available synthetic parotid hormone was found to have an activity indistinguishable from the isolated parotid hormone or the gene-expressed hormone.

Acinar Cell Source of the Parotid Hormone. Meanwhile, it was discovered that the acinar cells of the parotid glands are apparently the source of the parotid hormone.

Regulation of Insulin Secretion. Finally, preliminary research suggested the parotid hormone not only regulates dental structures but also influences insulin secretion by the pancreas.

Other Tissues/Organs. These systemic effects have yet to be followed up but may well include other glandular and/or avascular tissues. As Dr. Leonora stated in an undated letter, “I anticipate finding receptors [for the parotid hormone] in the odontoblasts in teeth, osteocytes in bone matrix, Islets of Langerhans. . . .”

Published Research

1. Leonora, J., McShan, W. H. & Meyer, R. K. (1956). Factors affecting extraction and recovery of follicle stimulating hormone from sheep pituitary glands. Proceedings of the Society for Experimental Biology and Medicine (92) 524-529.
2. Leonora, J., McShan, W. H., & Meyer, R. K. (1958). Separation of a luteinizing hormone fraction from sheep pituitary glands by use of ion exchange resin. Endocrinology (63) 867-888.
3. Meyer, R. K., Leonora, J., & McShan, W. H. (1959). Effectiveness of sheep FSH and LH preparations on spermiation in the frog. In A. Albert (Ed.), Human pituitary gonadotropins, pp. 326-328. Springfield, IL: Charles C. Thomas.
4. Meyer, R. K., Leonora, J., & McShan, W. H. (1959). Response of ovary, prostate and testes to human and sheep gonadotroic preparations. Effectiveness of human and sheep gonadotropic preparations on ovulation in immature rats. In A. Albert (Ed.), Human pituitary gonadotropins, pp. 329-334. Springfield, IL: Charles C Thomas.
5. Meyer, R. K., Leonora, J., & McShan, W. H. (1959). Effectiveness of human and sheep gonadotropic preparations on ovulation in immature rats. In A. Albert (Ed.), Human pituitary gonadotropins (pp. 335-338). Springfield, IL: Charles C Thomas.
6. Leonora, J., and Steinman, R. R. (1968). Evidence suggesting the existence of a hypothalamic parotid gland endocrine axis. Endocrinology (83). 807-815.
7. Steinman, R. R. and Leonora, J. (1969). Suppression of dental caries by chemical activation of the hypothalamic-parotid endocrine axis. Journal of Dental Research (48) S207, Abstr #666.
8. Sanchez, A., Steinman, R. R., & Leonora, J. (1970). Effect of diet on dentinal fluid movement in germfree and nongermfree rats. American Journal of Clinical Nutrition (23) 686-690.
9. Steinman, R. R., and Leonora, J. (1971). Relation between early changes in dentin and later dental caries. Journal of Dental Research (50)247, Abstr #780.
10. Steinman, R. R. and Leonora, J. (1971). Relationship of fluid transport through the dentin to the incidence of dental caries. Journal of Dental Research (50)6, Part 2:1536-1543.
11. Steinman, R. R., Leonora, J., Shiroma, E., & Dwyer, T. (1975). Lactic acid concentration in various tooth structures in rats maintained on either a cariogenic or noncariogenic diet. Journal of Dental Research (54)2. 419, Abstr.
12. Steinman, R. R., & Leonora, J. (1975). Effect of infusing selected chemical compounds on dentinal fluid movement in the rat. Journal of Dental Research (54)3. 567-569.
13. Steinman, R. R., & Leonora, J. (1975). Effect of selected dietary additives on the incidence of dental caries in the rat. Journal of Dental Research (54)3. 570-577.
14. Steinman, R. R., & Leonora, J. (1975). Acidogenic potential of cariogenic and noncariogenic diets in the rat. Journal of Dental Research (54)3. 578-580.
15. Steinman, R. R., & Leonora, J. (1977). Physiologic resistance to dental caries. Journal of the Missouri Dental Association (57)3. 14-21.
16. Steinman, R. R. & Leonora, J. & Tieche, J. M. (1979). Susceptibility to dental caries. Australian Dental Journal (24)4. 222-224.
17. Steinman, R. R., Leonora, J., & Singh, R. J. (1980). The effect of desalivation upon pulpal function and dental caries in rats. Journal of Dental Research (59)2. 176-185.
18. Tieche, J. M., Leonora, J., & Steinman, R. R. (1980). Isolation and partial characterization of a porcine parotid hormone that stimulates dentinal fluid transport. Endocrinology (106). 1994-2005.
19. Steinman, R. R. and Leonora, J. & Tieche, J. M. (1980). Susceptibility to dental caries: The pulp plays a major role. [Oral Health] (70). 43.
20. Tieche, J. M., Leonora, J., Berk, L. S., & Steinman, R. R. (1981). A radioimmunoassay procedure for quantitating parotid hormone. Biochemical and Biophysical Research Communications (98). 154-162.
21. Leonora, J., & Steinman, R. R. DFT: Its cariostatic effect. Teaching syllabus, 1981.
22. Leonora, J., Tieche, J. M., & Celestin, J. (1987). Physiological factors affecting the secretion of parotid hormone. American Journal of Endocrinology - Endocrinology and Metabolism (252). E477-E484.
23. Leonora, J. Tieche, J. M., and Cook, D. S. (1988). Evidence for the insulinotropic effect from rat parotid glands. Diabetes (37). 441-445.
24. Tieche, J. M., & Leonora, J. Biological and chemical evidence for the existence of a porcine hypothalamic parotid hormone-releasing factor. (1989). Biochemical and Biophysical Research Communications (159)3. 899-906.
25. Leonora, J., Tieche, J. M., & Steinman, R. R. (1992). The effect of dietary factors on intradentinal dye penetration in the rat. Archives of Oral Biology (37). 733-741.
26. Leonora, J., Tieche, J.M., & Steinman, R. R. (1993). Stimulation of intradentinal dye penetration by feeding in the rat. Archives of Oral Biology (38). 763-767.
27. Leonora, J. Tieche, J. M., & Steinman, R. R. (1993). Further evidence for a hypothalamus-parotid gland endocrine axis in the rat. Archives of Oral Biology (38). 911-916.
28. Tieche, J. M., Leonora, J. & Steinman, R. R. (1994). High-sucrose diet inhibits basal secretion of intradentinal dye penetration-stimulating parotid hormone in pigs. Journal of Applied Physiology (76). 218-222.
29. Tieche, J. M., & Leonora, J. (1995). Acute secretion of immunoreactive parotid hormone in response to different diets in the pig. Archives of Oral Biology (40)6. 559-565.
30. Leonora, J., Tjaderhane, L., & Tieche, J. M. Parotid (2002). Gland function and dentin apposition in rat molars. Journal of Dental Research (81)4. 259-264.
31. Leonora, J., Tjaderhane, L. and Tieche, J.M. (2002). Effect of dietary carbamyl phosphate on dentin apposition in rat molars. Archives of Oral Biology (47). 147-153.
32. Zhang, Q., Szalay, A. A., Tieche, J. M. Kyeyune-Nyombi, E., Sands, J. F., Oberg, K. C. and Leonora, J. (June 2005). Cloning and functional study of porcine parotid hormone, a novel proline-rich protein. Journal of Biological Chemistry (280)23. 22233-22244.

Research Abstracts

1. Leonora, J. and Steinman, R. R. Physiological observations of a new parotid hormone (PH). Proc. Inter. Union Physiol. Soc. (The Endocrine Society), Abstr. #781. Washington, DC., 1980.
2. Crane, M. G. and Leonora, J. Effects of medroxyprogesterone acetate and other steroids in combination with high sodium intake. Clin. Res. 18:167, 1970.
3. Leonora, J., and Crane, M. G. Effects of long-term administration of medroxyprogesterone acetate and other estrogens in the rat. Clin. Res. 18:169, 1970.
4. Wright, K. R., Weller, S. E., McMillan, P. J., Tieche, J. M., and Leonora, J. Sucrose-induced decrease in bone specific gravity in growing rats is inhibited by carbamyl phosphate. British Association of Clinical Anatomists with the American Association of Clinical Anatomists (3rd joint meeting), Cambridge, England, July 2000.

Contributions of Others

Throughout their research careers Drs. Steinman and Leonora acknowledged the contributions made by colleagues, students and fellow scientists. Following are illustrative quotations from their publications and related communications:

"The physiological significance of fluid flow across the dentin was first suggested by Gysi (1900) in an attempt to explain dentinal sensitivity." Leonora, J., Tieche, J. M., & Steinman, R. R. (1992). The effect of dietary factors on intradentinal dye penetration in the rat. Archives of Oral Biology (37). 733-741.

"The possibility of a circulation of dental fluid within dentinal tubules was suggested by Fish (1927) and supported experimentally by others (Bodecker and Lefkowitz, 1937; Lefkowitz, 1943)." Leonora, J., Tieche, J. M., & Steinman, R. R. (1992). The effect of dietary factors on intradentinal dye penetration in the rat. Archives of Oral Biology (37). 733-741.

"In 1934, Ogata . . . reported that bovine salivary glands exhibited a hormone-like activity and proposed a salivary gland endocrine theory. Similar studies were published by Takizawa and Hukusima . . . The hormone-like substance called parotin was isolated from bovine parotid glands and finally prepared in a 'homogeneous crystalline form' in 1949." Leonora, J., and Steinman, R. R. (1968). Evidence suggesting the existence of a hypothalamic parotid gland endocrine axis. Endocrinology (83). 807-815.

"Studies indicate that pulpal blood flow is affected by sympathetic adrenergic nerve fibers (Kim, Dorscher-Kim and Liu 1989). . ." Leonora, J., Tieche, J.M., & Steinman, R. R. (1993). Stimulation of intradentinal dye penetration by feeding in the rat. Archives of Oral Biology (38). 763-767.

"[N]europeptides and vasoactive agents have been identified in the pulp and/or shown to cause changes in pulpal blood flow when infused or applied topically. . . (Todoki 1988, Narhi 1989, Olgart, Edwall and Gazelius 1989, Liu et al 1990, Kim 1990, Casasco et al 1991)." Leonora, J., Tieche, J.M., & Steinman, R. R. (1993). Stimulation of intradentinal dye penetration by feeding in the rat. Archives of Oral Biology (38). 763-767.

"Vongasavan and Matthews (1992) directly measured the outward flow of fluid permeating through exposed dentin at the tips of fractured cat canine teeth." Leonora, J. Tieche, J. M., & Steinman, R. R. (1993). Further evidence for a hypothalamus-parotid gland endocrine axis in the rat. Archives of Oral Biology (38). 911-916.

References to Studies of Others

The following references represent additional studies by other researchers on the hormonal role of the parotid glands.

1. Dewey, M. M. (2005). A Histochemical and Biochemical Study of the Parotid Gland in Normal and Hypophysectomized Rats. American Journal of Anatomy (102)2. 243-271.
2. Fleming, H. S. The Effect of Parotin in Mice. (1960). Annals of the New York Academy of Sciences (85)1. 313-323.
3. Ginn, J. T. and Volker, J. F. (1942). Reduction of Growth in Desalivated Rats. Endocrinology (31). 282-294.
4. Gilda, J. E., and Keyes, P. H. (1947). Increased Dental Caries Activity in the Syrian Hamster Following Desalivation. Proc. Soc. Exper. Biol. & Med. (66). 28-41.
5. Iizuka, K., Togawa, K., and Konno, A. (1981). (1981). The Histogenesis of So-called Mixed Tumor Evaluating from Parotin Binding. European Archives of Oto-Rhino-Laryngology (233)2. 137-144.
6. Ito, Y. (1960). Parotin: A Salivary Gland Hormone. Annals of the New York Academy of Sciences (85)1. 228-312.
7. Kunio, T. and Toshio, S. (1971). Localization of Parotin in Bovine Parotid Gland, Demonstrated by the Immunohistochemical Method. Acta Histochemica et Cytochemica (4)1. 1-10.
8. Ogata, E., Suzuki, H., Shimazawa, E., Nakanowatari, K., and Asano, H. (1971). On the Hypocalcemic Effect in Rabbits of a Bovine Parotid Extract (Parotin). Endocrinol. Jpn. (18)3. 235-42.
9. Ogata, T. Internal Secretions of the Salivary Glands. (1935). Trans 9th Congress Far Eastern Association Tropical Medicine 2:709-715.
10. Shafer, W. G., and Muhler, J. C. (1960). Endocrine Influences Upon the Salivary Glands. Annals of the New York Academy of Sciences (85)1. 215-227.
11. Shaw, J. H., and Wesberger, D. (1949). Carious Lesions in Cotton Rat Molars. II. Effect of Removal of Principal Salivary Glands. Proc. Soc. Exper. Biology and Medicine (70). 103-107.
12. Sigheru, A. (1986). "Novel Peptide Having Dentinal Fluid Transport-Stimulating Activity." United States Patent 4745101 at http://www.freepatentsonline.com/4745101.html.
13. Suddick, R. P. Effect of Salivariadenectomy and Administration of Salivary Gland Homogenate upon the Reproductive Organs of the Female Rat. J. Dent. Res. 39:554-571, 1960.
14. Tateki, Y., Masao, K., and Masayoshi, K. Purification and Properties of Dentinal Fluid Transport Stimulating Substance from Bovine Parotid Glands. Chemical & Pharmaceutical Bulletin 34(3):1203-1211, 1986.
15. Tateki, Y., Masao, K., Masayoshi, K., Masaaki, Y., Masato, N., and Shigeru, A. (1986). Isolation and Amino Acid Sequence of Dentinal Fluid Transport-stimulating Peptide from Rat Parotid Glands. Chemical & Pharmaceutical Bulletin (34)9. 3803-3811.
16. Teruo, A. (1999). The Effect of Salivary Gland Hormone (Parotin) on Insulin Resistance. Ther. Res. (20)12. 3337-3340.

It is clear that Japanese and European as well as American scientists have long been involved in the parallel investigation of the hormonal role(s) of the salivary glands. None of them, however, have apparently pursued the critical role of the hypothalamus in this phenomenon. For further information, please see Clyde Roggenkamp (ed.) "Dentinal Fluid Transport". Loma Linda, CA: Loma Linda University Press, 2004.